Methods and compositions for rapid light-activated isolation and detection of analytes

ABSTRACT

The present invention relates to novel methods for isolating a target molecule from a sample suspected of containing the target molecule. The methods of the present invention utilize solid substrates as a means for capturing, separating, and releasing target molecules, such as chemical or biological compounds. The present invention is specifically directed to novel approaches for capturing, separating and releasing such target molecules.

RELATED APPLICATIONS

The present patent document claims the benefit of the filing date under35 U.S.C. §119(e) of Provisional U.S. Patent Application Ser. No.60/903,116, filed Feb. 23, 2007, which is hereby incorporated byreference.

FIELD

The present invention relates to the field of diagnostics. Moreparticularly, the invention disclosed herein is directed to methods,compositions, and kits for detecting nucleic acids, polynucleotides,pathogens, toxins, or other agents or factors that are desirablydetected or measured.

BACKGROUND

There is a great need to detect and quantify various molecular species,such as polynucleotides, polypeptides, carbohydrates, lipids, and smallmolecules. For example, current methods of detecting a polynucleotide,such as those associated with pathogens, pathogen infection, human genesassociated with diseases and disorders, altered physiology orphysiological conditions, genetically modified organisms (GMOs, i.e.,organisms with transgenic DNA), biowarfare agents, veterinaryapplications, and agricultural applications presently rely on complexmethods, such as the polymerase chain reaction (PCR), nucleic acidsequence-based amplification (NASBA), transcription-mediatedamplification (TMA), or branched DNA (bDNA). These methods requireskilled personnel and specialized equipment. Further, the methods aregenerally incapable of determining the presence or quantity ofpolynucleotides in crude cell and tissue extracts.

There are similar difficulties in the existing immunoassays fordetecting antigens. For example, antigens associated with bloodcoagulation disorders (e.g., F 1+2; Dade Behring, Bannockburn, Ill.),hepatitis infection (e.g., hepatitis B surface antigen; AbbottLaboratories, Abbott Park, Ill.), cancer-detection (e.g.,gastrointestinal stromal tumor-specific antigens; Ventana MedicalSystems, Inc., Tucson, Ariz.); acute pancreatitis (e.g., pancreaticelastase; Schebo-Biotech AG, Giessen, Germany), prostate cancer (e.g.,PSA; Beckman-Coulter, Inc., Fullerton, Calif.), and the like are allbased on multi-step ELISA immunoassays that require skilled personneland specialized equipment to run.

Accordingly, there is a great need for a convenient, fast and economicalmethod of detection, identification, and quantification of variousmolecules, such as polynucleotides and proteins, including polypeptides,peptides, and antigens. Reducing the complexity and increasing thereliability of such tests are among the features that would be desirablyimproved.

SUMMARY

The present invention relates to novel methods for isolating a targetmolecule from a sample suspected of containing the target molecule. Themethods of the present invention utilize solid substrates as a means forcapturing, separating, and releasing target molecules, such as chemicalor biological compounds. The present invention is specifically relatedto novel approaches for capturing, separating and releasing such targetmolecules.

In particular embodiments of the invention, the target molecule ofinterest is captured by indirectly immobilizing the target molecule to asolid support via the formation of one or more “intermediary” or“bridging” light reactive complex comprising two complementarypolynucleotide molecules, at least one of which is a nucleic acid analogpolynucleotide, such as a PNA polynucleotide, that is capable of forminga light reactive complex with the light reactive dye.

The formation of the light reactive complex occurs under highlyfavorable ambient light and temperature conditions, and may even occurin the dark. Surprisingly, the formation of the light reactive complexis not negatively impacted by even ambient light. In addition, thedisassociation of the light reactive complex from the solid substrate isfacilitated by similarly advantageous conditions of ambient light andtemperature, over time periods of brief duration (less than 2 hours,less than 1 hour, less than 30 minutes or less than 5 minutes), allowingthe method to be performed without equipment typically required, forexample, for nucleic acid hybridization and melting conditions used inpolymerase chain reaction methods or hybridization reaction methods,while still achieving an acceptable level of high degree of specificityand sensitivity. The methods of the present invention may therefore beadvantageously used, for example, in the field outside controlledlaboratory conditions, for the detection and analysis of a multiplicityof compounds or biological agents.

In one aspect, the present invention relates to methods for capturing atarget molecule, by combining

-   -   (i) a polynucleotide linked to target molecule,    -   (ii) a nucleic acid analog that is complementary to the        polynucleotide and is bound, directly or indirectly, to a solid        substrate, and    -   (iii) a light reactive dye,        to form a light reactive complex bound to the solid substrate,        which can then be separated, based on a property of the solid        substrate, as a unit, to thereby isolate the target        polynucleotide.

In one embodiment of the present invention, the methods of the presentinvention are used to isolate a target molecule that is apolynucleotide, such as a DNA or RNA molecule, by means of a directinteraction between a target polynucleotide and a capture nucleic acidanalog polynucleotide that is complementary to the target polynucleotideor a portion thereof and is immobilized on a solid substrate. Inaccordance with such embodiments, the methods of the invention comprisethe step of combining the following components:

-   -   (i) a sample suspected of containing the target polynucleotide;    -   (ii) a first nucleic acid analog immobilized to a solid        substrate, wherein the first nucleic acid analog is        complementary to a portion of the target polynucleotide; and    -   (iii) a first light reactive dye,        to form, if the target polynucleotide is present in the sample,        a light reactive complex comprising the target polynucleotide,        the first nucleic acid analog, and the light reactive dye,        followed by the step of separating, based on a property of the        solid substrate, the solid substrate and the light reactive        complex, as a unit, to thereby isolate the target polynucleotide        from the sample.

In another embodiment, the methods of the present invention are used toisolate a target molecule that is a polynucleotide, such as a DNA or RNAmolecule, by means of an indirect interaction between a target moleculeand a capture polynucleotide immobilized on a solid substrate. In theseembodiments, various intermediary constructs are used to link the targetmolecule with a capture polynucleotide immobilized to a solid support.The intermediary constructs are designed so as to provide at least onepair of complementary polynucleotides, at least one polynucleotide ofwhich is a nucleic acid analog, such as, for example, a PNApolynucleotide, a LNA polynucleotide, or morpholino polynucleotide, thatcan associate with a light reactive dye to form a light reactivecomplex.

In one embodiment, illustrating an indirect interaction between a targetmolecule and a capture nucleic acid analog polynucleotide immobilized ona solid substrate, the present invention relates to a method ofisolating a target molecule from a sample suspected of containing thetarget molecule, comprising the step of combining the following:

-   -   (i) a sample suspected of containing a target molecule;    -   (ii) a bound polynucleotide immobilized on a solid substrate;    -   (ii) a chimeric molecule comprising (1) a bridging        polynucleotide complementary to the bound polynucleotide,        and (2) a target component capable of binding to a portion of        the target molecule; and    -   (iv) a light reactive dye.

In this particular embodiment, the target component binds to the targetmolecule, if present, and the target component is linked to a bridgingpolynucleotide (the target component and the bridging polynucleotide arecollectively referred to as the “chimeric molecule”). The bridgingpolynucleotide is complementary to another polynucleotide that iscomplexed with the bound polynucleotide immobilized on the solidsubstrate. At least one of (a) the bridging polynucleotide of thechimeric molecule or (b) the bound polynucleotide immobilized on thesolid substrate, is a nucleic acid analog polynucleotide, which nucleicacid analog polynucleotide associates with its complementarypolynucleotide and the light reactive dye to form a light reactivecomplex immobilized on a solid substrate. Thus, the indirect interactionrequires at least one complementary pair of polynucleotides that canassociate with the light reactive dye to form a light reactive complex.

In yet another embodiment, illustrating another indirect interactionbetween a target molecule and a capture nucleic acid analogpolynucleotide immobilized on a solid substrate, the present inventionrelates to a method of isolating a target molecule from a samplesuspected of containing the target molecule, comprising the step ofcombining the following:

-   -   (i) a sample suspected of containing a target molecule,    -   (ii) a bound polynucleotide immobilized on a solid substrate,    -   (iii) an intermediary binding polynucleotide having a first        binding region complementary to the bound polynucleotide and a        second binding region,    -   (iv) a chimeric molecule comprising (1) a bridging        polynucleotide complementary to the second binding region of the        intermediary binding polynucleotide, and (2) a target component        capable of specifically binding to the target molecule, and    -   (v) a light reactive dye.

In this embodiment, the target component binds to the target molecule,if present, and the target component is linked to a bridgingpolynucleotide. In contrast to the embodiment described in the precedingparagraph (where the bridging polynucleotide forms a complex directlywith the bound polynucleotide immobilized on the solid substrate), thisembodiment contemplates the use of an intermediary bindingpolynucleotide, having a first binding region complementary to the boundpolynucleotide immobilized on the solid substrate, and a second bindingregion that is complementary to the bridging polynucleotide of thechimeric molecule to which the target component is linked. In thisconstruct, there are at least two regions of complementarity betweenseparate polynucleotides: the first region of complementarity is betweenthe bound polynucleotide and the first binding region of theintermediary binding polynucleotide; the second region ofcomplementarity is between the bridging polynucleotide and the secondbinding region of the intermediary binding polynucleotide. Either one orboth of these regions may form a light reactive complex with a lightreactive dye, provided that at least one of the complementary pair ofpolynucleotide is a nucleic acid analog, such as, for example, a PNApolynucleotide, an LNA polynucleotide, or a morpholino polynucleotide,which nucleic acid analog polynucleotide can associate with itscomplementary polynucleotide.

In another aspect, the methods of the present invention include the stepof separating the solid substrate (to which the target molecule isdirectly or indirectly attached) from the mixture, to thereby isolatethe target molecule. In typical applications, the target molecule willoriginate from a heterogeneous mixture, such as a blood sample, a watersample, a tissue sample, etc., which comprises various contaminantsmixed together with the target molecule of interest. The methods of thepresent invention are particularly useful in separating a targetmolecule from such a heterogeneous mixture. Thus, in one embodiment ofthe invention, the target molecule, immobilized to the solid substratevia at least one light reactive complex, is isolated, based on aproperty of the solid substrate, from a mixture, to thereby isolate thetarget polynucleotide from the sample.

In yet another aspect of the present invention, the target molecule,captured via formation of a light reactive complex between complementarypolynucleotides immobilized to a solid substrate, is released from thelight reactive complex.

In one embodiment, the present invention optionally includes the step ofexposing the light reactive complex, formed as described above, tolight, to thereby elute the target molecule from the solid substrateinto the eluant to provide a purified target molecule.

In yet another embodiment, the present invention relates to a method ofmaking a nucleic acid diagnostic kit comprising the step of combining,in a packaged combination, (i) an immobilized nucleic acid analog boundto a solid substrate, wherein the nucleic acid analog is complementaryto a portion of a target polynucleotide; and (ii) a light reactive dyein a container, wherein the light reactive dye is capable of forming alight reactive complex with the target polynucleotide and acomplementary nucleic acid analog.

In yet another embodiment, the invention relates to a method comprisingthe step of combining (i) a sample that may or may not contain a targetmolecule; (ii) an immobilized polynucleotide bound to a solid substrate;(iii) a bridging complex comprising (1) a polynucleotide portioncomplementary to the immobilized polynucleotide, wherein at least one ofthe immobilized polynucleotide and the polynucleotide portion of thebridging complex is a nucleic acid analog polynucleotide, and (2) atarget-specific portion capable of binding to the target molecule,wherein the bridging complex comprises one or more molecules complexedtogether; and (iv) a light reactive dye capable of forming a lightreactive complex with the immobilized polynucleotide and thecomplementary polynucleotide portion of the bridging complex.

In further embodiment, the invention relates to a method for making anucleic acid diagnostic kit comprising the step of combining, in apackaged combination, (i) an immobilized polynucleotide bound to a solidsubstrate; and (ii) a container comprising at least one of: a bridgingcomplex comprising (1) a polynucleotide portion complementary to theimmobilized polynucleotide, wherein at least one of the immobilizedpolynucleotide and the polynucleotide portion of the bridging complex isa nucleic acid analog polynucleotide, and (2) a target-specific portioncapable of binding to the target molecule, wherein the bridging complexcomprises one or more molecules complexed together; and a light reactivedye capable of forming a light reactive complex with the immobilizedpolynucleotide and the complementary polynucleotide portion of thebridging complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the percent change in fluorescence intensity as afunction of time exposed to light in mixtures that include 3,3′diethylthiacarbocyanine iodide dye, ncPNA, target polynucleotide, andone of a series of surfactants.

FIG. 2 depicts the percent change in fluorescence intensity as afunction of time exposed to light in mixtures that include 3,3′diethylthiacarbocyanine iodide dye, ncPNA, target polynucleotide, andone of a series of surfactants at 5.0% concentration by volume.

FIG. 3 depicts the percent change in fluorescence intensity as afunction of time exposed to light in mixtures that include 3,3′diethylthiacarbocyanine iodide dye, bacterial cells containing targetpolynucleotide, and one of a series of surfactants at indicatedconcentration by volume.

FIG. 4 depicts the percent change in fluorescence intensity as afunction of time exposed to light in mixtures that include 3,3′diethylthiacarbocyanine iodide dye, target polynucleotide, and one of aseries of nucleic acid analogs.

FIGS. 6A-B depict different schemes of capturing and detectingpolynucleotide targets, either on a solid substrate or in a liquid.

FIG. 6C-D depict means of detecting mutations using nucleic acidanalogs.

FIG. 7 depicts an agarose gel containing 3,3′-diethylthiacarbocyanineiodide dye in which a non-chiral PNA/target polynucleotide hybrid isresolved.

FIG. 8 depicts a series of single nucleotide polymorphisms detected bythe methods disclosed herein.

FIG. 9 depicts a series of two or more nucleotide polymorphisms detectedby the methods disclosed herein.

FIG. 10 depicts the change in fluorescence over time using differentsurfactants in a white plate under lower light intensity.

FIGS. 11A-D depict the change in fluorescence intensity as a function oftime exposed to light of the “non-chiral PNA:DNA+Dye” reactions and the“Dye Only” reactions using different microtiter plates.

FIG. 12 depicts the reduction in background noise signal from actualsignal using a modified phosphate buffer, altered light intensity with awhite plate.

FIG. 13 depicts the use of a bacterial permeabilization/lysis buffer ona gram positive bacterial and a gram negative bacterial sample withbacterial specific and non-specific nucleic acid analog probes.

FIG. 14 depicts the use of an altered bacterial permeabilization/lysisbuffer and quantitative detection of a bacterial target on a seriallydiluted culture of bacterial cells.

FIG. 15 depicts the reduction of background noise signal from a specificsignal by use of both Tween® 80 and methanol in the buffer compared tomethanol alone. Tests were done at reduced light intensities in a whiteplate.

FIGS. 16A-B is, respectively, a schematic of a P/TP as a “detector”attached to an antibody and an agarose gel shift assay.

FIG. 17 is a comparison of a “cocktail” of 12mer PNAs and a “cocktail”of 17mer PNAs in reactions with MTB (A) or human (B) genomic DNA in thepresence of the dye, exposed to light over time.

FIG. 18 depicts typical dye absorbance change vs. time data for thevarying amounts of MTB genomic DNA.

FIG. 19 depicts initial absorbance change vs. time data for differentconcentrations of MTB genomic DNA

FIG. 20 shows an MTB DNA dose response curve from initial rate datademonstrating a linear relationship between analyte concentration andinitial slope value.

FIG. 21 depicts dye absorbance change vs. time data for differentconcentrations of MTB genomic DNA samples

FIG. 22 depicts an MTB DNA dose response curve from time to absorbancedate where the relation between time to absorbance and analyteconcentration is not typically linear

FIG. 23 depicts a polyacrylamide gel showing dark areas in the gel wherephotobleaching occurred (samples containing PNA-RNA duplexes).

DETAILED DESCRIPTION

The present invention provides methods, compositions and kits fordetermining the presence or amount of a target molecule by using nucleicacid analogs and a dye in the context of a reaction mixture that has acharacteristic optical property. The target molecule can be anymacromolecule or small molecule, as further detailed below. Even whenthe target molecule is a polynucleotide, the nucleic acid analogs usedin the present invention may or may not include a sequence that iscomplementary to a segment or moiety of the target molecule. The presentinvention, generally speaking, relates to the presence in a reactionmixture of a hybrid nucleic acid molecule that includes the nucleic acidanalog if the target molecule is present; and if so, then the opticalproperty of the reaction mixture changes, thereby indicating thepresence of the target molecule.

I. GENERAL TECHNIQUES

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, protein kinetics, and mass spectroscopy, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL (2d ed., Cold Spring Harbor Press 2000); CELL BIOLOGY: ALABORATORY NOTEBOOK (J. E. Cellis, ed., Academic Press 1998); ANIMALCELL CULTURE (R. I. Freshney, ed., 1987); METHODS IN ENZYMOLOGY (aseries of volumes directed at enzymology protocols that is published byAcademic Press, Inc.); HANDBOOK OF EXPERIMENTAL IMMUNOLOGY (D. M. Weirand C. C. Blackwell, eds.); PCR: THE POLYMERASE CHAIN REACTION (Mulliset al., eds., 1994); and the like. Furthermore, procedures employingcommercially available assay kits and reagents typically are usedaccording to manufacturer-defined protocols, unless otherwise noted.

II. DEFINITIONS

The term “target molecule” generally refers to a molecule having anucleic acid sequence or an antigenic determinant or a carbohydrate thatis detected using the methods, compositions, or kits disclosed herein. Atarget molecule can be a macromolecule or a small molecule as thoseterms are used in the art. In particular, a macromolecule is apolynucleotide, a polypeptide, a carbohydrate, a lipid, or a combinationof one or more of these. As a general rule, the molecular mass of amacromolecule is at least about 300 Daltons and can be millions ofDaltons. A small molecule is an organic compound having a molecularweight of up to about 300 Daltons.

The term “target nucleic acid sequence” refers to the nucleic acidsequence of a target polynucleotide that hybridizes to a nucleic acidanalog or the nucleic acid sequence of an intermediary polynucleotide ina sequence specific manner, for the purpose of detecting the targetpolynucleotide using the methods, compositions or kits disclosed herein.All or part of the target polynucleotide or intermediary polynucleotidewhich is at least partially hybridized with the target polynucleotidemay form a hybrid with a nucleic acid analog by sequence-specifichybridization, albeit some mismatch may exist depending on theconditions of the reaction mixture. The binding could be by way ofWatson-Crick hybridization or sequence specific binding modes yetundescribed. The target nucleic acid sequence may be of any length. Incertain instances, the target nucleic acid sequence is preferably lessthan about 1000 bases, less than about 500 bases, less than about 100bases, less than about 40 bases, or less than about 24 bases. In otherembodiments, the target nucleic acid sequence is greater than about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 12, about14, about 16, about 18, about 20, about 25, about 30, about 35, about40, about 45, or about 50 bases in length. In yet other embodiments, thetarget nucleic acid sequence is preferably greater than about 4 basesand less than about 24 bases in length. In certain embodiments, thetarget nucleic acid sequence is about 4, about 6, about 8, about 10,about 12, about 14, about 16, about 18, about 20, about 22, or about 24bases in length. The target nucleic acid sequence may include a proteincoding sequence and/or a non-coding sequence (e.g., intergenic spacersequences regulatory sequences, introns, and the like).

The term “polynucleotide” refers to a polymeric form of nucleotides ornucleotide analogs of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof, or mixtures thereof.Polynucleotides may be single-stranded, double-stranded,triple-stranded, or multi-stranded to yet greater degrees. The followingare non-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,armored RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, nucleic acid probes, primers,amplified DNA, and synthesized DNA. A polynucleotide may containmodified bases, including those that include, without limitation, amethylation, deamination, thiolation, and/or acetylation. The sequenceof nucleotides of a polynucleotide may be interrupted by non-nucleotidecomponents, and may include one or more nucleic acid analogs. Apolynucleotide may be further modified before or after polymerization,such as by conjugation with a labeling component. The polynucleotide maybe an amplified region of a longer sequence of nucleotides. Apolynucleotide may be a peptide nucleic acid (PNA) or a chiral PNA or anachiral PNA, among other nucleic acid analogs.

The term “target polynucleotide” refers to a polynucleotide thatincludes a target nucleic acid sequence. The target polynucleotide maybe of any length. In certain instances, the target polynucleotide ispreferably less than about 1000 bases, less than about 500 bases, lessthan about 100 bases, less than about 40 bases, or less than about 24bases. In other embodiments, the target polynucleotide is preferablygreater than about 8, about 9, about 10, about 12, about 14, about 16,about 18, about 20, about 25, about 30, about 35, about 40, about 45,about 50, about 75, about 100, about 150, about 200, about 250, about300, about 350, about 400, about 450, about 500, or about 1000 bases inlength. In yet other embodiments, the target polynucleotide ispreferably greater than about 20 bases and less than about 1000 bases inlength; more preferably, greater than about 20 and less than about 500;even more preferably, greater than about 20 and less than about 400bases in length. In certain embodiments, the target polynucleotide isabout 50, about 100, about 150, about 200, about 250, about 300, about350, about 400, about 450, about 500, or about 1000 bases in length.Other lengths are also contemplated. For example, the targetpolynucleotide may be from 2 to 5 kb in length.

The term “intermediary polynucleotide” refers to a polynucleotide usedin an indirect hybridization method for detecting a targetpolynucleotide. The intermediary polynucleotide includes a portion thatis complementary to a target polynucleotide and another portion that iscomplementary to a nucleic acid or a nucleic acid analog. One example ofsuch intermediary polynucleotide is shown in FIG. 6A. In anotherembodiment, the intermediary polynucleotide may be employed as a pair.An example of such intermediary polynucleotide is shown in FIG. 6B. Eachof the pair of intermediary polynucleotides includes, in the followingorder, (1) a first segment that is complementary to contiguous segments,respectively, of the target polynucleotide, (2) a second segment that isthe complement of the analogous portion of the other intermediarypolynucleotide, and (3) a third segment that is complementary tocontiguous segments of a nucleic acid, which may or may not be a nucleicacid analog.

“Armored RNA™” refers to an RNA that is ribonuclease resistant due tothe encapsidation of the RNA by bacteriophage proteins. “Armored RNA™”is further described, for example, in U.S. Pat. Nos. 6,399,307;6,214,982; 5,939,262; 5,919,625; and 5,677,124.

The term “nucleic acid analog” refers to any molecule that is describedin part by a sequence of bases, as is commonly done for DNA or RNA,which molecule has one or more bases that differ from conventionalguanine, thymine, adenosine, cytosine, or uracil, and/or having one ormore differences from the conventional phosphoribose of an RNA backboneor the conventional phosphodeoxyribose of a DNA backbone at one or morebases. The nucleic acid analog is preferably greater than about 4 basesin length and less than about 24 bases in length, excluding linkers,amino acids and labels. In other embodiments, the nucleic acid analogmay be from about 5 to about 100, from about 8 to about 60, or fromabout 10 to about 20 bases in length. In another embodiment, the nucleicacid analog is about 6, about 8 about 10, about 12, about 14, about 18,about 22, about 26, about 30, about 35, about 40, or about 45 bases inlength, excluding linkers, amino acids and labels. In other embodiments,the target nucleic acid can be at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 12, at least 14,at least 15, at least 18, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50 bases in length.Nucleic acid analogs can be chimeric by having a specific type ofnucleic acid analog nucleoside in combination with another nucleic acidanalog nucleoside, and/or one or more conventional DNA nucleosides orRNA nucleosides.

Exemplary phosphorous modifications useful in creating nucleic acidanalogs include chiral phosphorothioate (bridging and non-bridging),phosphorodithioate, chiral methyl phosphonate, chiral phosphoramidate,chiral phosphate trimester, chiral boranophosphate, and chiralphosphoroselenoate. Exemplary linkage modifications includemethylenemethylimino (MMI), 3′-amide, 3′ achiral phosphoramidate, 3′archiral methylene phosphonate, thioformacetal, and thioethyl ethermodifications. Exemplary sugar modifications include 2′-fluoro,2′-O-methyl, 2′-O-(3-amino)propyl, 2′-O-(2-methoxy)ethyl,2′-O-2-(N,N-dimethylaminooxy)ethyl (DMAOE),2′-O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl (DMAEOE), and2′-O—N,N-dimethylacetamidyl. Classes of analog nucleotides having sugarmodifications include N-morpholinophosphordiamidate (Morpholinos);hexose nucleic acid (HNA); threose nucleic acid (TNA), such as thosedisclosed in Chaput et al., AMER. CHEM. SOC., 125:856-857 (2003);cyclohexene nucleic acid (CeNA); locked nucleic acid (LNA), havingmethylene bridges between the 2′-O and 4′-C on the ribofuranose ring ofsome or all individual nucleotides of a polynucleotide (which methylenebridges function to restrict the flexibility of the polynucleotide andare associated with enhanced stability and hybridizationcharacteristics), such as those disclosed in TRENDS IN BIOTECHNOLOGY21:74-81 (2003); and tricycle-deoxyribose nucleic acid (tcDNA)modifications. Preferred base modifications include5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl,7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl,phenoxazinyl, phenoxazinyl-G-clamp, 2,6-diamino purine, and 2,6-diaminothiouracil. A preferred connection modification is anα-deoxyribofuranosyl. Preferred sugar replacement modifications includeproduction of a peptide nucleic acid (PNA) or a chiral PNA or an achiralPNA. Other exemplary nucleic acid analogs include sequence-specific DNAbinding minor groove ligands, such as polyamides (containing imidazole(Im), pyrrole (Py), and hydroxypyrrole (Hp)). Nucleic acid analogs canbe chimeric, and have multiple different modifications, and can includenon-nucleic acid analogs, such as linkers, as are known in the art.Polynucleotides that include analog nucleotides are described as tosequence with respect to its non-modified analog as identified in theaccompanying Sequence Listing, with further description of which, if notall, included nucleotides are modified.

The term “photochemical reaction” refers to a reaction that can occurwhen electromagnetic radiation interacts with matter and initiates theproduction of new chemical species. Absorption of electromagneticradiation, typically in the region of the electromagnetic spectrum whichranges from approximately 180 nanometers in the ultraviolet to 800nanometers in the near infrared, initiate electronic transitions in theabsorbing species and result in a temporary change in its electronicstructure. This electronically excited species may reemit the energyabsorbed via radiationless decay, fluorescent emission, orphosphorescent emission resulting in no change to the original absorber.Alternatively, the electronically activated species can undergo anirreversible electronic change creating a new product molecule ormolecules. Also, the electronically excited species can interact with asecond molecule with different chemical structure in the sample causingchanges in that molecule's electronic structure which in turn can causereversible or irreversible changes to the second molecule. Products ofthese photochemically induced reactions can in turn react with otherchemically distinct molecules in the sample to initiate other chemicalreactions.

The term “reaction mixture” refers to the mixture of at least thefollowing when the polynucleotide is the complement of a target nucleicacid sequence of the target molecule, which, in this case, would be atarget polynucleotide: (1) a target polynucleotide; (2) apolynucleotide; and (3) a dye. Alternatively, when the polynucleotideand its complement are unrelated to the target molecule, and instead areattached to a target binding component as part of the reporter complex,the reaction mixture comprises: (1) a target molecule; (2) a targetbinding component; and (3) a dye.

The term “peptide nucleic acid,” or “PNA,” includes any nucleic acidanalog in which the deoxyribose phosphate backbone of a nucleic acid hasbeen replaced by a synthetic peptide-like backbone, including, forexample, n-(2-amino-ethyl)-glycine units, such as, without limitation,those disclosed in U.S. Pat. Nos. 5,786,461; 6,357,163; 6,107,470;5,773,571; 6,441,130, 6,451,968; 6,228,982; 5,641,625; 5,766,855;5,736,336; 5,719,262; 5,714,331; 5,719,262; and 6,414,112. The purineand pyrimidine bases may be attached by any covalent linkage, including,for example, methylene carbonyl linkages. As used herein, PNA moleculescan have additional atoms between the PNA backbone and nucleobase. Theseanalogs include, for example, D-lysine chains, cyclic structures, suchas cyclopentane or pyrrolidine rings, and/or chiral substituents,including PNA molecules described in U.S. Pat. No. 6,403,763, U.S.Patent Application US 2003/0162699, and U.S. Patent Application US2003/0157500. The PNA backbone may include substitutions or extensionsin the peptide backbone. PNAs may include peptide-based nucleic acidmimics (PENAMS), such as those disclosed, for example, in U.S. Pat. No.5,705,333, atoms having unusual chiral centers, such as D-chiral centersand quasi-chiral centers, and atom substitutions in the PNA backbone.

The term “chiral PNA” or “cPNA” refers to a chiral PNA molecule in whichat least a portion of the peptide backbone has been modified to includea proline or modified proline side-chain that includes the backbonenitrogen and α-carbon. Non-limiting examples of chiral PNA moleculesinclude those that are disclosed at, for example, U.S. Pat. No.6,403,763, U.S. Patent Applications US 2003/0162699 and US 2003/0157500.

The term “achiral PNA” or “non-chiral PNA” or “ncPNA” refers to a PNAmolecule in which no portion of the peptide backbone has been modifiedto include a proline or modified proline side chain that includes thebackbone nitrogen and α-carbon.

The term “non-PNA nucleic acid analog” refers to a nucleic acid analogin which the backbone is not made up of n-(2-amino-ethyl)-glycinesubunits.

The term “locked nucleic acid” or “LNA” refers to a bicyclic nucleicacid in which at least one ribonucleoside is linked between the2′-oxygen and the 4′-carbon with a methylene group. Non-limitingexamples of LNAs are disclosed in TRENDS IN BIOTECHNOLOGY 71:74-81(2003).

The term “morpholino nucleic acid” or “MNA” refers to a nucleic acidanalog in which each backbone monomer is a substituted or unsubstitutedsix-membered morpholino ring. The morpholino rings are linked bynon-ionic phosphorodiamidate linkages. Non-limiting examples of MNAsinclude those described in U.S. Pat. No. 5,034,506.

The term “threose nucleic acid” or “TNA” refers to a nucleic acid inwhich the sugar-phosphate backbone is a four-carbon sugar threose inplace of the five-carbon sugar ribose.

The term “metal linked nucleic acid” or “MLNA” refers to a nucleic acidsequence in which at least a portion of the ribose phosphate backbone ismodified with a transition metal. Non-limiting examples of MLNAs includethose MLNAs disclosed at the website of the Wilker Research Group,Purdue University website.

The terms “nucleic acid analog/polynucleotide hybrid” and“polynucleotide/nucleic acid analog hybrid” and “P/TP hybrid” aresynonymous and refer to a nucleic acid analog and target polynucleotidehybridized in a sequence-specific manner. Non-limiting examples ofnucleic acid analog/polynucleotide hybrids include nucleic acidanalog/polynucleotide duplexes and triplexes.

The terms “PNA/polynucleotide hybrid” and “polynucleotide/PNA hybrid”are synonymous and refer to a PNA and polynucleotide hybridized in asequence-specific manner. Non-limiting examples of PNA/polynucleotidehybrids include PNA/polynucleotide duplexes and triplexes. The PNA maybe chiral or non-chiral.

By “complementary” it is meant that a single-stranded nucleic acidanalog has the ability to bind a polynucleotide in a base-specificmanner. The nucleic acid analog may be synthesized to bind a targetpolynucleotide, such as a full-length polynucleotide strand or a partthereof. A nucleic acid analog that is “complementary” may have one ormore single base-pair mismatches, additions, and/or deletions, but isstill capable of hybridizing to the target polynucleotide under theselected hybridization or association conditions. In one embodiment,complementary sequences may hybridize through Watson-Crick base pairing(A-T or A-U and C-G or alternatively pairing with inosine). In a furtherembodiment, complementary sequences may hybridize through Hoogstein basepairing. In alternative embodiment, complementary sequences mayhybridize through formation of a unique dye-PNA-DNA composite. In otherwords, the dye may function as an accelerator for DNA duplex formation.

The term “hybrid” refers to an association between a dye, nucleic acidanalog and a target polynucleotide in a manner that forms a complex andpermits detecting a change in an optical property when specific bindingoccurs versus when specific binding does not occur. It is not known whatphysical or structural relationship occurs between the dye, nucleic acidanalog and the target polypeptide in the hybrid. Without being bound bya particular theory, formation of a duplex, triplex, Watson-Crick basepairing, Hoogstein base pairing, or any other yet undefined binding orassociation is contemplated herein. As such, the term “hybrid” is notlimited to any particular physical or structural relationship betweenthe elements of the hybrid.

By “exactly complementary”, it is meant that the single-stranded nucleicacid analog has the ability to hybridize to a target nucleic acidsequence without base mismatches. A nucleic acid analog is not exactlycomplementary to a target polynucleotide if there is a single base-pairmismatch between the nucleic acid analog and the target polynucleotide.

The term “rate” refers to a change (e.g., of a property of a compositionor compound). A rate may be described in terms of a specific rateconstant. A rate may be determined by making measurements over a periodof time. A rate may be described by making measurements, determined bymeasurements at two different time points in a process or by makingmeasurements at least three, at least four, or at least five,timepoints. A rate may be determined based on a single measurement and aknown quantity, such as a previously known or calculated quantity. Arate may be expressed in quantitative or qualitative terms (e.g., achange is “fast” or “slow”). A rate may be determined by comparing aproperty or compound to a reference value, or by observation of changesin a given property or compound over time, using standard methods.

As used herein, the term “relative rate” refers to the rate of oneprocess compared to the rate of another process. A “relative rate” maybe approximate (e.g., the rate of one process may be “faster” or“slower” than the rate of another process) or quantitative (e.g.,comparing measured rate constants of two processes).

As used herein, the term “dye” refers to a first compound that has ameasurable optical property or that may be converted to a secondcompound with a measurable optical property. Preferred dyes includethose where the measurable optical property thereof differs incomparison to that of the second compound. Measurable optical propertiesinclude, but are not limited to color, absorbance, percenttransmittance, fluorescence, reflectance, chemiluminescence, andinfrared (IR) spectrum measurements. The dye may exhibit the opticalproperty under certain conditions, such as binding or forming a complexor otherwise being in contact with a target polynucleotide/nucleic acidanalog hybrid or, or not binding or forming a complex or otherwise beingin contact with a target polynucleotide/nucleic acid analog hybrid.

The term “light reactive dye” means a dye that reacts to light exposure,such that when the light reactive dye is associated in a complex withcomplementary polynucleotides, at least one of which is a nucleic acidanalog, such as a PNA molecule, the light reactive dye confers aproperty on the complex that, in response to exposure of the complex tolight, results in the disassociation of the complex and a correspondingchange in optical property of the light reactive dye. In someembodiments, the complementary polynucleotides of the complex maycomprise nucleic acid analog and a standard nucleic acid. In otherembodiments, the complex may comprise two nucleic acid analogs.Complexes formed between the light reactive dye and complementarypolynucleotides comprising at least one nucleic acid analogpolynucleotide are referred to herein as “light reactive complexes.”

A “hybrid catalyst” refers to a hybrid molecule that is capable ofpromoting the photodegradation of a dye. The following examples areusefully employed in the present invention: nucleic acid-nucleic acidhybrids, nucleic acid analog-nucleic acid hybrids, nucleic acidanalog-nucleic acid analog hybrids. The present invention establishesreaction conditions by which the nucleic acid-nucleic acid hybridspresent in any cell lysate, for example, contribute minimally to thecatalytic function assigned to any of these hybrids. The same conditionsthat minimize the ability of the nucleic acid-nucleic acid hybrids tochange an optical property also tend to potentiate the activity of thenucleic acid-nucleic acid analog hybrids.

“Sample” refers to a liquid sample of any type (e.g., blood, serum,water, urine, fecal matter, sputum, or lysate or extract of a solidsample), a solid sample of any type (e.g., cells, food, ice, dirt,grain, or material acquired from a surface), an airborne sample of anytype, and/or a material embedded in a gel material and/or anysolid-phase material, such as agarose, acrylamide, or gelatin.

The term “pathogen” refers to any agent causing a disease, disorderand/or pathological condition and/or symptoms. By way of example, thepathogen may be an organism (or its associated toxin) found in nature,or created in a laboratory, that causes disease in or development of apathological condition or symptom in, incapacitates, debilitates and/orkills an organism. Pathogens include, but are not limited to, viruses,bacteria, fungi, eukaryotes, and/or prokaryotes; and may function asbiological weapons agents, or vectors of infectious diseases; and mayspread via water, as in water-borne pathogens, food, as in foodpathogens, air or direct skin contact.

The term “biological weapons agent” refers to any organism (or itsassociated toxin) found in nature or created in the laboratory that isused for the primary purpose of causing disease in, incapacitating, orkilling another living organism. Examples of biological weapons agentsinclude, but are not limited to, pathogenic bacteria, fungi, protozoa,rickettsiae, and viruses. The target of a biological weapons agentincludes any of humans, animals, and plants, as well as sub-populationsthereof.

As used herein, the term “infection” refers to the presence of apathogen in or on a host. The infection may be dormant or virulent. Inone embodiment, the presence of the pathogen is indicated by analteration in host polynucleotide and/or polypeptide expression.Infection may occur through such routes including, but not limited to,airborne droplets, direct contact, animal or insect vectors, andcontaminated food or drink.

As used herein, the term “host-response polynucleotide” refers to apolynucleotide that is altered, or a polynucleotide for which theexpression is altered, in a host in response to a stimulus, such asinfection, and/or contact by a pathogen.

The term “host” as used herein refers to humans, animals, and plants.The animal may be a mammal. Examples of mammals include, non-humanprimates, farm animals, sport animals, mice, and rats. Examples ofplants include, but are not limited to, dicot or monocot agriculturalcrops.

As used herein, the term “alkyl,” “alkenyl,” and “alkynyl” refer tostraight-chain, branched-chain and cyclic monovalent substituents, andcan be substituted or unsubstituted. Examples include methyl, ethyl,isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and thelike. Typically, the alkyl, alkenyl and alkynyl substituents containC₁₋₁₀ (alkyl) or C₂₋₁₀ (alkenyl or alkynyl). Preferably they containC₁₋₆ (lower alkyl) or C₂₋₆ (lower alkenyl or lower alkynyl). Examples ofalkyl groups include propyl, tert-butyl, and cycloalkyls such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptylgroups. Examples of alkenyl groups include allyl, crotyl, 2-pentenyl,3-hexenyl, 2-cyclopentenyl, 2-cyclohexenyl, 2-cyclopentenylmethyl, and2-cyclohexenylmethyl groups.

As used herein, the terms “heteroalkyl,” “heteroalkenyl,” and“heteroalkynyl” are similarly defined but may contain one or more O, Sor N heteroatoms or combinations thereof within the backbone residue;collectively, the aforementioned terms having the “hetero-” prefix arereferred to as “hetero forms.”

As used herein, “acyl” encompasses the definitions of alkyl, alkenyl,alkynyl, each of which is coupled to an additional residue through acarbonyl group. Heteroacyl includes the related heteroforms.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety such as phenyl or naphthyl; “heteroaryl” refers tomonocyclic or fused bicyclic ring systems containing one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits inclusion of 5-membered rings as well as 6-membered rings. Thus,typical aryl/heteroaryl systems include pyridyl, pyrimidyl, indolyl,benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl,benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl,and the like. Because tautomers are theoretically possible, phthalimidois also considered aromatic. Any monocyclic or fused ring bicyclicsystem that has the characteristics of aromaticity in terms of electrondistribution throughout the ring system is included in this definition.Typically, the ring systems contain 5- to 12-ring-member atoms.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aryl andheteroaryl systems that are coupled to another residue through a carbonchain, including those carbon chains that are substituted orunsubstituted, saturated or unsaturated, typically having one to eightcarbon atoms, including hetero forms thereof. These carbon chains mayalso include a carbonyl group, thus making them able to providesubstituents as an acyl or heteroacyl moiety.

In general, any alkyl, alkenyl, alkynyl, acyl, or aryl group containedin a substituent may itself optionally be substituted by additionalsubstituents. The nature of these substituents is similar to thoserecited with regard to the primary substituents themselves. Thus, wherean embodiment of a substituent is alkyl, this alkyl may optionally besubstituted by the remaining substituents listed as substituents wherethis makes chemical sense, and where this does not undermine the sizelimit of alkyl per se; e.g., alkyl substituted by alkyl or by alkenylwould simply extend the upper limit of carbon atoms for theseembodiments. However, alkyl substituted by aryl, amino, alkoxy, and thelike would be included.

As used herein, the term “halogen” and “halo” are used interchangeablyand refer to one or more substituents including fluorine, chlorine,bromine, iodine, and astatine.

Examples of substituted hydroxyl and thiol groups include substitutedalkyloxy or alkylthio (e.g., C₁₋₁₀ alkyl), such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, asubstituted arylalkyloxy or arylalkylthio (e.g., phenyl-C₁₋₄ alkyl,benzyl, or phenethyl). Where there are two adjacent hydroxyl or thiolsubstituents, the heteroatoms may be connected via an alkylene groupsuch as O(CH₂)_(n)O and S(CH₂)_(n)S (where n=1-5).

Examples of substituted hydroxyl groups also include optionallysubstituted C₂₋₄ alkanoyl (e.g., acetyl, propionyl, butyryl orisobutyryl), C₁₋₄ alkylsulfonyl (e.g., methanesulfonyl orethanesulfonyl) and a substituted aromatic and heteroaryl carbonylgroup, including benzoyl and pyridinecarbonyl.

Substituents on substituted amino groups may bind to each other to forma cyclic amino group (e.g., 5- to 6-membered cyclic amino, etc., such astetrahydropyrrole, piperazine, piperidine, pyrrolidine, morpholine,thiomorpholine, pyrrole or imidazole). The cyclic amino group may have asubstituent, and examples of the substituents include halogen, nitro,cyano, hydroxy group, thiol group, amino group, carboxyl group, anoptionally halogenated C₁₋₄ alkyl, an optionally halogenated C₁₋₄ alkoxy(e.g., methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, etc.), C₂₋₄alkanoyl (e.g., acetyl or propionyl), and C₁₋₄ alkylsulfonyl (e.g.,methanesulfonyl or ethanesulfonyl).

An amino group may also be substituted once or twice (to form asecondary or tertiary amine) with a group such as an optionallysubstituted alkyl group including C₁₋₁₀ alkyl (e.g., methyl or ethylpropyl); an optionally substituted alkenyl group, such as allyl, crotyl,2-pentenyl, 3-hexenyl, and the like, or an optionally substitutedcycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl or cycloheptyl. In certain cases, these groups are C₁₋₆alkyl, C₂₋₆ alkenyl, or cycloalkyl groups. The amine group may also beoptionally substituted with an aromatic or heteroaromatic group, aralkyl(e.g., phenyl C₁₋₄ alkyl) or heteroalkyl, for example, phenyl, pyridine,phenylmethyl (benzyl), phenethyl, pyridinylmethyl, or pyridinylmethyl.The heteroaromatic group may be a 5- or 6-membered ring containing 1-4heteroatoms.

An amino group may be substituted with an optionally substituted C₂₋₄alkanoyl (e.g., acetyl, propionyl, butyryl, and isobutyryl), or a C₁₋₄alkylsulfonyl (e.g., methanesulfonyl or ethanesulfonyl), or a carbonyl-or sulfonyl-substituted aromatic or heteroaromatic ring (e.g.,benzenesulfonyl, benzoyl, pyridinesulfonyl and pyridinecarbonyl). Theheteroaromatics are as defined above.

Examples of carbonyl groups, sulfinyl groups, or sulfonyl groups includesubstituted or unsubstituted forms of such groups formed from varioushydrocarbyls, such as alkyl, alkenyl and 5- to 6-membered monocyclicaromatic group (e.g., phenyl and pyridyl), as defined above.

The term “salt” is meant to include salts of the active compounds fromany acid or base known in the art, as appropriate to the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acid addition salts include (1) any halogen; (2) thosederived from an inorganic acid, such as hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acid, or the like; andthose derived from relatively nontoxic organic acids, such as acetic,propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids, including arginate and the like, and salts of organicacids, including glucuronic or galacturonic acid and the like (see, forexample, S. M. Berge et al., J. PHARMA. SCI. 66:1-19 (1977)). Certainspecific compounds of the present invention contain both basic andacidic functionalities that allow the compounds to be converted intoeither base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents. Otherwise, however, the salts are equivalent to theparent form of the compound for the purposes of the present invention.

As used herein, the term “target binding component” refers to a moleculecapable of interacting with a target molecule. Target binding componentshaving limited cross-reactivity are generally preferred. In certainembodiments, suitable target binding components include, for example:lectins; receptors; antibodies, including monoclonal antibodies,polyclonal antibodies, and derivatives or analogs thereof, includingwithout limitation, Fv fragments, single chain Fv (scFv) fragments, Fab′fragments, F(ab′)₂ fragments, single domain antibodies, camelizedantibodies and fragments thereof, humanized antibodies and fragmentsthereof, and multivalent versions of the foregoing. Multivalent versionsof target binding components include without limitation: monospecific orbispecific antibodies, such as disulfide stabilized Fv fragments, scFvtandems ((scFV)₂ fragments), diabodies, tribodies or tetrabodies, whichtypically are covalently linked or otherwise stabilized (i.e., leucinezipper or helix stabilized) scFv fragments. Other binding reagentsinclude, for example, template imprinted materials (such as those ofU.S. Pat. No. 6,131,580), and organic or inorganic binding elements. Incertain embodiments, a target binding component specifically interactswith a single identifying unit of the target molecule, such as anepitope or a sugar or a ligand. In other embodiments, a target bindingcomponent may interact with several structurally related epitopes orsugars or ligands.

The term “reporter complex” refers to a first reporter nucleotidesequence, a second reporter nucleotide sequence, and a dye. The firstreporter nucleotide sequence and second reporter nucleotide sequence canbe covalently bonded together, or not covalently bonded together.

The term “modified target binding component” refers to a target bindingcomponent that is covalently modified by at least one component of thereporter complex.

The term “target binding complex” refers to the modified target bindingcomponent and the remaining components of the reporter complex.

The term “bridging complex” refers to one or more molecules that linkthe solid substrate to a target specific portion that binds to a desiredtarget molecule.

The term “antibody” refers to an immunoglobulin, derivatives thereofthat maintain specific binding ability, and proteins having a bindingdomain that is homologous or largely homologous to an immunoglobulinbinding domain. These proteins may be derived from natural sources, orpartly or wholly synthetically produced. An antibody may be monoclonalor polyclonal. The antibody may be a member of any immunoglobulin class,including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Incertain embodiments, antibodies used with the methods and compositionsdescribed herein are derivatives of the IgG class.

The term “antibody fragment” refers to any derivative of an antibodythat is less than full-length. In certain embodiments, the antibodyfragment retains at least a significant portion of the full-lengthantibody's specific binding ability. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFvdiabody, and Fd fragments. The antibody fragment may be produced by anymeans. For instance, the antibody fragment may be enzymatically orchemically produced by fragmentation of an intact antibody, it may berecombinantly produced from a gene encoding the partial antibodysequence, or it may be wholly or partially produced synthetically. Theantibody fragment may optionally be a single chain antibody fragment.Alternatively, the fragment may comprise multiple chains that are linkedtogether, for instance, by disulfide linkages. The fragment may alsooptionally be a multimolecular complex. A functional antibody fragmentwill typically comprise at least about 50 amino acids and more typicallywill comprise at least about 200 amino acids.

As used herein, the term “array” refers to a set of target bindingcomponents immobilized onto one or more substrates so that each targetbinding component is at a known location. Alternatively, the set oftarget binding components may be in solution, respectively in differentreceptacles of a microtiter dish, and therefore, located at knownlocations. In one embodiment, a set of target binding components isimmobilized onto a surface in a spatially addressable manner so thateach individual target binding component is located at different andidentifiable location on the substrate.

The term “camelized antibody” refers to an antibody or variant thereofthat has been modified to increase its solubility and/or reduceaggregation or precipitation, similar to that found in a camelid.Camelids produce heavy-chain antibodies consisting only of a pair ofheavy chains wherein the antigen binding site comprises the N-terminalvariable region or VEH (variable domain of a heavy chain antibody). TheVHH domain comprises an increased number of hydrophilic amino acidresidues that enhance the solubility of a VHH domain as compared to a VHregion from noncamelid antibodies. Camelization of an antibody orvariant thereof preferably involves replacing one or more amino acidresidues of a non-camelid antibody with corresponding amino residuesfrom a camelid antibody.

The term “chemical handle” refers to a component that may be attached toa target binding complex as described herein so as to facilitate theisolation, immobilization, identification, detection and/or increasedsolubility of the target binding complex. Suitable chemical handlesinclude, for example, a polypeptide, a polynucleotide, a carbohydrate, apolymer, or a chemical moiety, and combinations or variants thereof.

The term “diabodies” refers to dimeric scFvs. The components ofdiabodies preferably have shorter peptide linkers than most scFvs andthey show a preference for associating as dimers. The term diabody isintended to encompass both bivalent (i.e., a dimer of two scFvs havingthe same specificity) and bispecific (i.e., a dimer of two scFvs havingdifferent specificities) molecules. Methods for preparing diabodies areknown in the art. See, e.g., EP 404097 and WO 93/11161.

As used herein, the term “epitope” refers to a physical structure on amolecule that interacts with a target binding component. In exemplaryembodiments, epitope refers to a desired region on a target moleculethat specifically interacts with a target binding component.

The term “Fab” refers to an antibody fragment that is substantiallyequivalent to that obtained by (1) digestion of immunoglobulin(typically IgG) with the enzyme papain or (2) reduction of the disulfidebridge or bridges joining the two heavy chain pieces in the F(ab′)₂fragment. The heavy chain segment of the Fab fragment is the Fd piece.Such fragments may be enzymatically or chemically produced byfragmentation of an intact antibody, recombinantly produced from a geneencoding the partial antibody sequence, or wholly or partiallysynthetically produced. Methods for preparing Fab fragments are known inthe art. See, e.g., Tijssen, PRACTICE AND THEORY OF E NZYME IMMUNOASSAYS(Elsevier, Amsterdam, 1985).

The term “F(ab′)₂” refers to an antibody fragment that is substantiallyequivalent to a fragment obtained by digestion of an immunoglobulin(typically IgG) with the enzyme pepsin at pH 4.4. Such fragments may beenzymatically or chemically produced by fragmentation of an intactantibody, recombinantly produced from a gene encoding the partialantibody sequence, or wholly or partially synthetically produced.

The term “Fv” refers to an antibody fragment that consists of one VH andone VL domain held together by noncovalent interactions. The term “dsFv”is used herein to refer to an Fv with an engineered intermoleculardisulfide bond to stabilize the VH-VL pair. Methods for preparing Fvfragments are known in the art. See, e.g., Moore et al., U.S. Pat. No.4,462,334; Hochman et al., BIOCHEMISTRY 12:1130 (1973); Sharon et al.,BIOCHEMISTRY 15:1591 (1976); and Ehrilch et al., U.S. Pat. No.4,355,023.

The term “immunogen” refers to compounds that are used to elicit animmune response in a human or an animal, and is used as such herein.Many techniques used to produce a desired target binding component, suchas the phage display methods described below, do not rely wholly, oreven in part, on immunizations. Nevertheless, these methods usecompounds containing an “epitope,” as defined above, to select for andclonally expand a population of target binding components specific tothe “epitope.” These in vitro methods mimic the selection and clonalexpansion of immune cells in vivo. Therefore, the compounds containingthe “epitope” that is used to clonally expand a desired population ofphage and the like in vitro are embraced within the definition of“immunogens.” Similarly, the terms “hapten” and “carrier” have specificmeaning in relation to standard immunization protocols. In that context,and as used herein, a “hapten” is preferably a small molecule thatcontains an epitope, but is incapable of serving as an immunogen byitself. Therefore, to elicit an immune response to the hapten, thehapten is preferably conjugated with a larger carrier, such as bovineserum albumin or keyhole limpet hemocyanin, to produce an immunogen. Animmune response would recognize the epitope on the hapten, but would notrecognize any epitopes that may be on the carrier.

In the in vitro methods described herein for preparing the desiredbinding reagents, traditional “haptens” and “carriers” typically havetheir counterpart in epitope-containing compounds affixed to suitablesubstrates or surfaces, such as beads and tissue culture plates.

The terms “single-chain Fvs” and “scFvs” refers to recombinant antibodyfragments consisting of only the variable light chain (VL) and variableheavy chain (VH) covalently connected to one another by a polypeptidelinker. Either VL or VH may be the NH₂-terminal domain. The polypeptidelinker may be of variable length and composition so long as the twovariable domains are bridged without function-defeating stericinterference. In exemplary embodiments, the linkers are comprisedprimarily of stretches of glycine and serine residues with some glutamicacid or lysine residues interspersed for solubility. Methods forpreparing scFvs are known in the art. See, e.g., PCT/TJS/87/02208 andU.S. Pat. No. 4,704,692.

The term “single domain antibody” or Fd refers to an antibody fragmentcomprising a VH domain that interacts with a given antigen. An Fd doesnot contain a VL domain, but may contain other antigen-binding domainsknown to exist in antibodies, such as the kappa and lambda domains.Methods for preparing Fds are known in the art. See, e.g., Ward et al.,NATURE 341:644-646 (1989) and EP 0368684.

The term “single chain antibody” refers to an antibody fragment thatcomprises variable regions of the light and heavy chains joined by aflexible linker moiety. Methods for preparing single chain antibodiesare known in the art. See, for example, U.S. Pat. No. 4,946,778 toLadner et al.

The term “triabody” refers to trivalent constructs comprising 3 scFv's,and thus comprising 3 variable domains (see, e.g., Iliades et al., FEBSLETT. 409(3):437-41 (1997)). This term also refers to molecules thatcomprise three variable domains having the same specificity, or threevariable domains wherein two or more of the variable domains havedifferent specificities.

The term “tetrabody” refers to engineered antibody constructs comprisingfour variable domains (see, e.g., Pack et al., J. MOL. BIOL. 246:28-34(1995) and Coloma and Morrison, NAT. BIOTECHNOL. 15:159-63 (1997)). Thisterm also refers to molecules that comprise four variable domains havingthe same specificity, or four variable domains wherein two or more ofthe variable domains have different specificities.

The term “VH” refers to a heavy chain variable region of an antibody.

The term “VL” refers to a light chain variable region of an antibody.

The term “optical property” refers to an intrinsic property of amaterial that can be observed when the material interacts withelectromagnetic radiation in the region of the electromagnetic spectrumthat ranges from approximately 180 nanometers in the ultraviolet to 40micrometers in the infrared. Observable optical properties include, butare not limited to absorption of specific wavelengths of electromagneticradiation, or absorption of specific wavelengths of electromagneticradiation followed by emission at other specific wavelengths. Measuringsuch optical properties is well known in the art and usescommercially-available UV-VIS spectrometers and fluorophotometers. Inthe particular case of chiral molecules, the absorption of polarizedelectromagnetic radiation may also be measured by techniques such asPolarimetry and Circular Dichroism.

The term “observing” means detecting a change in a property or value,either directly or indirectly, by means of visual observation,instrumentation, or receipt of data. As used herein, the terms“observing” and “detecting” are synonymous.

Observable optical properties can also include chemical changes thathappen to the sample when it absorbs electromagnetic radiation.Absorption of electromagnetic radiation may initiate an electronicrearrangement in the absorbing species and result in a change in itschemical reactivity or structure, or this electronically-activatedspecies may interact with another molecule in the sample causing itsstructure or reactivity to change. In either of these cases, changescould occur in the absorption or emission spectra of the sample. Also,such changes could result in the appearance or disappearance of one ofthe materials in the reaction mixture. Such changes could therefore bemeasured by any of the aforementioned photometric methods.

III. FURTHER SUMMARIZATION OF THE EMBODIMENTS

The present invention relates in a first embodiment to a method ofdetecting a target polynucleotide in a sample. Preferably, the methodincludes producing a reaction mixture comprising a sample that may ormay not contain a target polynucleotide, a nucleic acid or a nucleicacid analog that is immobilized to a solid substrate and which iscomplementary to a portion of the target polynucleotide, and a lightreactive dye capable of forming a light reactive complex with the targetpolynucleotide, if present in the sample, and a complementary nucleicacid analog. Preferably, the nucleic acid or the nucleic acid analog isat least partially complementary to a segment of the targetpolynucleotide. The method may further include the step of isolating thesolid substrate based on a property of the solid substrate. The methodmay further comprise the step of washing the solid substrate with a washsolution comprising the light reactive dye. Another step of the methodmay involve exposing the reaction mixture, including the solid substrateto a light under conditions sufficient to elute the targetpolynucleotide from the solid substrate. The light also may serve toactivate the reaction that causes the change in the optical property ofthe reaction mixture. Yet another aspect of the method involvesobserving the optical property of the reaction mixture at least onceafter exposure to the light. Preferably, the reaction mixture has anoptical property that changes in response to the light exposure if thefirst nucleic acid or first nucleic acid analog and the targetpolynucleotide are present therein, and wherein the dye is the compoundof formula (I), or a salt or ester thereof, as described elsewhere inthis specification. The present invention further provides forcorrelating the detecting of the target polynucleotide with theresultant change in the optical property of the reaction mixture.

Although applicants are not asserting any particular theory or mechanismby which the method set forth here works, evidence has been gainedregarding the chemical reaction of oxidation that appears to beassociated with at least one of the dyes usefully employed in thediagnostic test. Accordingly, the change in the optical property notedas a reporter of certain recognition-based activity of nucleic acids andbinding pair type molecules correlates with a chemical change in thedye. Other mechanisms may be in play in the alternative or in addition.Suffice to say that when the aforementioned components of a reactionmixture are combined followed by a light exposure for activation, theoptical property is commonly seen with a wide number of cyanine dyes.

A part of the present invention is the observation of the reactionmixture to determine whether the optical property has changed.Preferably, one will observe the reaction mixture at least twice.

In one embodiment, the optical property includes a first opticalproperty that diminishes after exposure to the light and a secondoptical property that increases after exposure to the light. Typically,the property that diminishes is inherent to the reaction mixture, andreliably occurs to the extent that the reaction mixture includes anucleic acid hybrid and dye, where the nucleic acid hybrid is composedof any pair-wise combination of a nucleic acids and nucleic acidanalogs. Upon the diminishing optical property, the reaction mixture,contained in a vessel, further comprises a substance that delivers thesecond optical property; alternatively, the second optical property iscontributed by a substance applied to the vessel itself, for example.The substance that contributes this second optical property may or maynot be a soluble dye.

In another embodiment, the reaction mixture includes a detergent. Thedetergent has multiple functions, including stabilizing the dye,apparently, and lysing cells for study. Where a sample includes intactcells prior to being in contact with the detergent, the cell lyses andrenders its genetic material, for example, available for study.

The reaction mixture in a further embodiment is directed at a reactionmixture that includes an achiral peptide nucleic acid. A similarlypreferred alternative are those reaction mixtures that include a chiralpeptide nucleic acid.

In another embodiment, the length of the target polynucleotide isgreater than about 50 bases.

In yet another embodiment, the reaction mixture further comprises asecond nucleic acid, wherein at least a portion of the second nucleicacid is complementary to a portion of the first nucleic acid that is notcomplementary to the target polynucleotide. This reaction mixturefurther comprises a third nucleic acid, and wherein one portion of thethird nucleic acid is complementary to a portion of the targetpolynucleotide that is not complementary to the first nucleic acid andwherein another portion of the third nucleic acid is complementary to aportion of the second nucleic acid that is not complementary to thefirst nucleic acid. Preferably, the first part and the second part ofthe nucleic acid analog do not overlap.

The nucleic acid analog used in the context of the present invention isgreater than about 4 bases in length and less than about 24 bases inlength. In another embodiment, the nucleic acid analog is about 12nucleic acid bases in length. In a further embodiment, the nucleic acidanalog is 17 nucleic acid bases in length.

The present method also includes immobilizing the target polynucleotideand the nucleic acid analog on a solid substrate. In a preferred aspect,the nucleic acid analog is attached to a solid substrate.

In additional embodiment, the present method includes immobilizing andreleasing the target polynucleotide and the nucleic acid analog from asolid substrate, as described in more detail in Section V below. In oneaspect, the nucleic acid analog is attached to a solid substrate.

In one embodiment, the dye is a compound of formula (I), wherein R₁, R₂,and R₃ are hydrogen or hydrophobic alkyls, R₄ through R₁₃ are hydrogen,and Y is sulfur. In a second embodiment, the dye is of formula (I),wherein n is 1. More preferably, the dye is of formula (I), wherein n is1; and wherein Y is sulfur or —CR₁₂═CR₁₃—. Even more preferably, the dyeis of formula (I), wherein n is 1; wherein Y is sulfur or —CR₁₂═CR₁₃—;and wherein R₁ and R₂ are each independently selected from the groupconsisting of alkyl and alkenyl.

Yet another embodiment relates to a method of detecting a targetpolynucleotide in a sample, which includes producing a reaction mixturecomprising the sample, a nucleic acid analog that is complementary to atarget nucleic acid sequence of the target polynucleotide, and a dye;exposing the reaction mixture to a light; and observing the absorbanceof the reaction mixture at least once. Preferably, the reaction mixturehas an absorbance that changes if the target polynucleotide and thenucleic acid analog form a hybrid therein. Also preferably, the dye isthe compound of formula (I), as set forth elsewhere in thisspecification. This embodiment further includes correlating thedetecting of the target polynucleotide with the resultant change in theoptical property of the reaction mixture. What is observed is thepotential change in the optical property of the reaction mixture, whichcorrelates with a chemical change in the dye.

The present invention also relates to a composition that includes asurfactant and a dye according to formula (I) or formula (II) or formula(III), or a salt thereof, as set forth elsewhere here.

The inventive composition can further include a nucleic acid analog; ortarget polynucleotide.

Yet another embodiment is a kit for detecting a target polynucleotide,which includes one or more nucleic acid analogs at least partiallycomplementary to a target nucleic acid sequence of said targetpolynucleotide; one or more dyes; one or more surfactants; andinstructions that relate to the method set forth herein above.

Another embodiment is a reporter complex that includes a firstpolynucleotide, a second polynucleotide, and a dye, wherein the firstpolynucleotide and the second polynucleotide form a hybrid and thereporter complex has an optical property that changes in response toexposure to a light stimulus. The hybrid used in the reporter isattached to a target binding component, which target binding componentis selected from the group consisting of an antibody or fragmentthereof, a lectin, or a receptor, among other selective binding agents.For example, aptamers, molecular imprints, and avimers may all be usedin the context of the present invention. In a particularly preferreduse, the polypeptide and the nucleic acid analog are immobilized on asolid substrate. In another embodiment, a second polynucleotide may be anucleic acid analog. The second nucleic acid analog may be the same asor different from the first nucleic acid analog. The second nucleic acidanalog may be bound to a solid substrate or may be in solution not bebound to a solid substrate. Alternatively, the reporter may be a singlepolynucleotide that forms a hairpin.

The present invention also relates to a method for detecting a targetmolecule in a sample, in which the target molecule and a target bindingcomponent bind one another with substantial specificity includingcombining the sample, the target binding component, a firstpolynucleotide, and a second polynucleotide in a reaction mixture in avessel. Preferably, the first polynucleotide and second polynucleotideform a hybrid and are in contact with the target binding component. Alsopreferably, at least one of the target binding component, firstpolynucleotide, and second polynucleotide, or the target molecule areattached to a solid surface. The method also includes washing thereaction mixture; and combining the reaction mixture components that areimmobilized on the solid substrate with dye, whereupon the reactionmixture has an optical property that changes if the sample includes thetarget molecule and it and the target binding component bind oneanother. A further aspect includes exposing the reaction mixture tolight; and observing the optical property of the reaction mixture atleast once. Again, this method is used with one of the dyes disclosedelsewhere, namely a compound of formula (I).

Another aspect of the present invention is a catalytic hybrid thatincludes two polynucleotides or a polynucleotide and a nucleic acidanalog or two nucleic acid analogs that together form the hybrid, or asingle polynucleotide or nucleic acid analog or chimeric structure thatforms an internally complementary hairpin structure, wherein the hybridcatalyses a chemical reaction of a dye upon exposure to a lightstimulus. The dye that the catalytic hybrid acts upon has beenwell-described here, namely a compound of formula (I).

For each of the methods disclosed here, the quantity of the targetpolynucleotide is determined by comparing the observed optical propertyas compared to a reference.

IV. METHODS OF DETECTING POLYNUCLEOTIDES

The present invention relates to methods, compositions and kits fordetermining the presence or amount of a target polynucleotide having atarget nucleic acid sequence.

In one embodiment, (i) a sample for testing for the presence or amountof a target polynucleotide, (ii) a polynucleotide that binds a targetnucleic acid sequence of the target polynucleotide in asequence-specific manner, and (iii) a dye are combined to produce areaction mixture that has an observable optical property that can changeover time. If the target polynucleotide is present, then it and thepolynucleotide will form a hybrid (referred to herein as the “P/TP”hybrid; if the polynucleotide is a nucleic acid analog, then the hybridis referred to as a NAA/TP). The rate of change in the optical propertyof the mixture is preferably different in the presence and absence ofthe P/TP hybrid. In one aspect of the invention, a light stimulus isapplied to the mixture. The light stimulus when so applied may serve toactivate the reaction that results in a change in the optical property.The change in the optical property of the reaction mixture (i.e., thedye disappearing) does not occur in the absence of the light stimulus;the activation provided by the light stimulus is correlated with anincrease in the rate of change of the optical property of the reactionmixture.

The rate of change in the optical property of the mixture is preferablycompared to a reference value characteristic of the rate of change ofthe optical property in a similar mixture containing a known amount(which can be a zero amount) of the P/TP hybrid to determine a relativerate of change in the optical property. The relative rate of change inthe optical property of the mixture is correlated with the presence oramount of the target polynucleotide in a sample to determine thepresence or amount of target polynucleotide in the sample.

An alternative method substitutes the comparison of rates of change toan observation of the optical property after exposure of the reactionmixture to a light stimulus. One can conclude that the targetpolynucleotide is present to the extent that an observable change in theoptical property occurs after the light stimulus exposure. One can alsoapproximate the amount of the target polynucleotide that is present inthe sample by comparing the optical property of the light-stimulusexposed reaction mixture after or upon a minimal or certain incubationtime to a standard chart that displays the observable optical propertyas it will appear by the minimal or at the certain time. The standardchart is preferably generated by conducting the detection method withsamples having known amounts of the target polynucleotide, memorializingthe standard resultant state of optical property by, for example, takingcolor or black and white photographs of the mixtures after the minimalor at the certain time of incubation, and assembling the standard chartusing the memorialized record of the state of the optical property ofthe mixture. Of course, one must take care that the memorialization asto quality (color) and/or quantity (intensity) is substantiallyaccurate. Alternatively, the values of the standard chart can begenerated by an equation.

A reference value can be a value characteristic of a property of acomposition or compound having a known characteristic. For example, invarious embodiments, a reference value can be determined using a mixturethat does not contain an P/TP hybrid; contains a known amount of an P/TPhybrid; or is a reaction mixture from which one or more components(e.g., a nucleic acid analog, a target polynucleotide, or a dye) hasbeen omitted, each of which are “controls” of the inventive method. Insome instances, the reference value may be a known quantity or ameasured quantity. Further nonlimiting examples of reference valuesinclude a value characteristic of an optical property of a mixture thathas not been exposed to light stimulus, or, in an alternativeembodiment, an optical property of a mixture that has been exposed tolight stimulus. The aforementioned examples are for illustration and arenot intended to limit the methods, and other examples will be apparentto the practitioner guided by this disclosure. It will be appreciatedthat a reference value may be, but need not necessarily be, empiricallydetermined. For example, if it is known that the optical properties of acomposition containing a dye do not change, or change minimally, in theabsence of the P/TP hybrid, the reference value may be calculated orinferred and not measured. The reference value may be a constant.Although in some cases it may be convenient to assay a “control” sampleconcurrently with test samples, in certain embodiments of the method itis not necessary to do so. A reference value can be determined at onetime point, and the value recorded for comparison at later time pointsas in the standard chart noted above. It will be understood that theaforementioned examples are for illustration and not limitation.

In one aspect, the reference value is characteristic of the rate ofchange in the optical property of a similar mixture containing no P/TPhybrid. In one embodiment, the reference value may be characterized bythe optical property contributed by the dye prior to the combination ofall the components in the mixture. Alternatively, the reference valuecan be external to the mixture, as in a color affixed to the vessel inwhich the reaction mixture is located. In another embodiment, thereference value may be characterized by the optical product of thereaction. For embodiments in which the mixture is exposed to lightstimulus, the reference value may be characteristic of the opticalproperty of the dye or mixture containing the dye prior to applying thelight stimulus. It is not a requirement of the present invention thatthe reference value is necessarily determined by preparing one or morecontrol samples that are included in separate mixtures and otherwisetreated substantially identically as the experimental sample. Inaddition, it is understood that the reference value may be a constant.

The present invention is further directed to methods in which (i) asample containing, or not containing, or possibly containing a targetpolynucleotide, (ii) a nucleic acid analog that binds a target nucleicacid sequence of the target polynucleotide in a sequence-specificmanner, and (iii) a dye are preferably combined to form a mixture. Alight stimulus is preferably applied and the intensity of an opticalproperty of the mixture is observed. In one embodiment, the decrease inintensity of the mixture is correlated to the presence or amount oftarget polynucleotide in the sample. Alternative methods for observingthe reaction include detection of the product of the photochemicalreaction or detection of a previously hidden, masked or quenchedcomponent.

In certain embodiments, a dye preferably exhibits an initial colorchange when in the presence of a P/TP hybrid. Following the initialcolor change, the color of the mixture decreases until the mixturebecomes substantially clear (i.e., lacks or nearly lacks color). Therate of the change in the optical property corresponding to the changeof the mixture from the presence of a color to less color or thesubstantial lacking of color is preferably measured. The rate of changein the optical property can thereby be determined.

In still other methods, a sample and a non-PNA nucleic acid that iscomplementary to a target nucleic acid sequence in a targetpolynucleotide are combined with a dye to form a mixture. The mixturehas a different optical property in the presence or absence of a P/TPhybrid. A change in the optical property of the mixture correlates tothe presence of a target nucleic acid sequence.

In another embodiment of the invention, the time required to reach aspecific change in an optical property is measured, and the percentchange thereof is preferably calculated. Alternatively, the change inthe optical property at a specific time point can also be measured, ormerely observed where an approximate determination will suffice. Forexample, if the amount of target polynucleotide in a sample is known,the measured optical property at a specific time can be employed inassessing the relative amount of the target polynucleotide in a secondsample of unknown target polypeptide content. By observing the opticalproperty change at the specific time and comparing it to thecharacteristic of the known sample at the same point in the reaction theobserver can conclude that the second sample contains a greater, aboutthe same, or lesser amount or concentration of the target polypeptide asexists in the known sample. The amount or quantity of the target nucleicacid could thus be determined in a binary fashion, and the amount aswell, albeit to an approximation or a comparative value of lesser ormore.

A. Dyes

A group of related dyes are preferably used in the methods hereof todetect the presence or amount of a target polynucleotide.

In one embodiment, the preferred dye is a compound that is representedby the formula (I), or a salt or betaine thereof:

In formula (I), independently at each occurrence,R₁ and R₂ are each independently selected from hydrogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, acyl,heteroacyl, heteroaryl, hydroxyl, alkoxy, carbonyl, sulfinyl, sulfonyl,and amino groups;R₃ is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, acyl,heteroacyl, heteroaryl, aryl, alkyl, heteroaryalkyl, hydroxyl, alkoxy,halo, carbonyl, sulfinyl, sulfonyl, and amino groups;R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, and R₁₃ are each independentlyselected from the group consisting of hydrogen, halogen, alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, acyl,heteroacyl, heteroaryl, aryl, alkyl, heteroaryalkyl, hydroxyl, alkoxy,carbonyl, sulfinyl, sulfonyl; whereinn is 0, 1, 2, 3, 4, or 5; andY is selected from the group consisting of —C₁₂═CR₁₃—, sulfur, nitrogen,and oxygen. In one embodiment, a dye is the compound of formula (I),wherein Y is —C₁₂═CR₁₃—, sulfur, or oxygen. In yet another embodiment, adye is the compound of formula (I), wherein Y is sulfur; or wherein Y isC₁₂═CR₁₃—; or wherein Y is oxygen.

In another embodiment, the dye is preferably a compound that isrepresented by the formula (II), or a salt or ester thereof:

In formula (II), independently at each occurrence, R₁ and R₂ are eachindependently selected from C₁-C₆ alkyl, C₂-C₆ alkenyl, and C₂-C₆alkynyl;

R₃ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, and C₂-C₆ alkynyl, C₆-C₁₀ aryl, hydroxyl, alkoxy, carbonyl,sulfinyl, sulfonyl, and amino groups;

n is 1 or 2;

R₄ and R₉ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, hydroxyl,alkoxy, halo, carbonyl, sulfinyl, sulfonyl, and amino groups.

In a further embodiment, the dye is a compound that is represented bythe formula (III), or a salt or ester thereof:

In formula (III), independently at each occurrence:

R₁ and R₂ are each independently selected from C₁-C₆ alkyl and C₂-C₆alkenyl;

R₃ is selected from the group consisting of hydrogen and methyl;

n is 1 or 2.

In yet other embodiments, the dye is preferably selected from thefollowing compounds:

where X- is an anion. More preferably, the anion is a halogen; yet morepreferably, the anion is iodide.

In one embodiment of formula (I, II, and III), n is 0.

In another embodiment of formula (I, II, and III), n is 1.

In another embodiment of formula (I, II, and III), n is 2.

In yet another embodiment of formula (I, II, and III), n is 3.

In one embodiment of formula (I, II, and III), R₁ and R₂ are eachindependently selected from group consisting of hydrogen, alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, andheteroaryl.

In another embodiment of formula (I, II, and III), R₁ and R₂ are eachindependently selected from group consisting of alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, and heteroalkynyl.

In another embodiment of formula (I, II, and III), R₁ and R₂ are eachindependently selected from group consisting of alkyl, alkenyl, andalkynyl.

In another embodiment of formula (I, II, and III), R₁ and R₂ are eachindependently alkyl.

In one embodiment of formula (I, II, and III), R₃ is selected from thegroup consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, and heteroalkynyl.

In one embodiment of formula (I, II, and III), R₃ is selected from thegroup consisting of alkyl, alkenyl, and alkynyl.

In one embodiment of formula (I, II, and III), R₃ is alkyl.

The compounds in the following list are within the scope of formulas(I-III. Nevertheless, because they did not perform optimally under theconditions described in Example 4, the dyes of this list are excludedfrom use in accordance with one aspect of the invention:3,3′-Diethylthiacyanine; 3-Ethyl-9-methyl-3′-(3-sulfatobutyl)thiacarbocyanine; 3,3′-Dimethyloxacarbocyanine;3-Carboxymethyl-3′,9-diethyl-5,5′-dimethylthiacarbocyanine;3,3′-Diethylthiadicarbocyanine; 3,3′-Diethylthiatricarbocyanine;3,3′-Diethyloxacarbocyanine; 3,3′-Diethyloxadicarbocyanine;3,3′-Dipropylthiadicarbocyanine; 3,3′-Dipropyloxacarbocyanine;3,3′-Dihexyloxacarbocyanine; 3,3′-Diethyl-2,2′-oxathiacarbocyanine;1,1′-Diethyl-2,2′-cyanine; 1,1′-Diethyl-2,4′-cyanine;1,1′-Diethyl-4,4′-carbocyanine;1,1′-Diethyl-3,3,3′,3′-tetramethylindocarbocyanine;1,1′-Dipropyl-3,3,3′,3′-tetramethylindocarbocyanine;[5-[2-(3-Ethyl-3H-benzothiazol-2-ylidene)-ethylidene]-4-oxo-2-thioxo-thiazolidin-3-yl]-aceticacid;1-Butyl-2-[3-(1-butyl-1H-benzo[cd]indol-2-ylidene)-propenyl]-benzo[cd]indolium;5,6-Dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-benzimidazol-2-ylidene)-propenyl]-1,3-diethyl-3H-benzimidazolium;1,3,3-Trimethyl-2-(2-[2-phenylsulfanyl-3-[2-(1,3,3-trimethyl-1,3-dihydro-indol-2-ylidene)-ethylidene]-cyclohex-1-enyl]-vinyl)-3H-indolium;4,5,4′,5′-Dibenzo-3,3′-diethyl-9-methyl-thiacarbocyanine; and Thiazoleorange.

Additionally, in accordance with another aspect of the invention3,3′-diethylthiacarbocyanine iodide is also excluded from use withrespect to the present invention.

Suitable dyes can be identified using any of a variety of screeningmethods. The suitability of a dye is a function of its ability tocontribute an optical property to the reaction mixture, as describedhere, which optical property changes in response to and preferably, tothe extent of the presence of a hybrid between either (1) a nucleic acidanalog (“NAA”) and a nucleic acid (“NA”), i.e., an NAA/NA; or (2)between two complementary NAs, i.e., a NA/NA. By way of example and notlimitation, a NAA/NA, or a NA/NA, or a P/TP (collectively referred to asthe “hybrid”) is prepared for identifying suitable dyes. Preferably, thecombination includes other components and is maintained in conditions topromote formation of the hybrid, using such components and conditionsthat are well known in the art. Suitable conditions and components aredescribed herein. The candidate dye is then preferably added; morepreferably, the candidate dye is added to separate aliquots of thecombination, such that the dye is present in the separate aliquots atvarying concentrations. Yet more preferably, a detergent, or a detergentand an alcohol, is added. The order of addition is not critical; thecomponents can be added in any order. Once the reaction mixture isformed, a light stimulus is preferably applied, although notnecessarily. The rate of change in the optical property over time isthen determined. This rate is compared to a reference valuecharacteristic of the rate of change in optical property of the reactionmixture in the absence of the hybrid. Example 4 illustrates one methodfor screening dyes for suitability for the inventive methods disclosedherein.

In certain embodiments, the reference value is characteristic of theabsence of the target polynucleotide or the presence of the targetpolynucleotide, which can be single-stranded or double-stranded. Inother embodiments, the reference value is characteristic of a non-zeroconcentration of the target polynucleotide. Preferably, the referencevalues employed include those characteristic of a zero and at least onenon-zero concentration of the target polynucleotide, respectively.

In certain embodiments, the change in optical property can be the lossof intensity of the optical property. Dyes that contribute an opticalproperty to the reaction mixture are particularly preferred, where themixture exhibits a different rate of change in optical property overtime compared to a reference value. The relative rate of change in theoptical property of the mixture is correlated with the presence oramount of the target polynucleotide.

Alternatively, dyes that contribute an optical property to the reactionmixture, where the reaction mixture changes color in the presence of anNAA/NA hybrid, are preferably selected. Preferably, the nucleic acidanalog used in the context of a color changing optical property is anon-PNA variety. Such nucleic acid analogs include, without limitation,an LNA, TNA, MLNA, and a morpholino nucleic acid.

B. Designing Nucleic Acid Analog Sequences

For use in the present invention, nucleic acid analogs may be designedto be complementary, but possibly including some mismatched bases,additions or deletions, or be exactly complementary to a nucleic acidtarget.

The sequence of the nucleic acid analogs or target polynucleotides maybe designed in a variety of ways. By way of example and not limitation,the nucleic acid analogs or their respective complementarypolynucleotides may be designed to have sequences based on known primersused for PCR-based amplification and detection of specific targetsequences. The nucleic acid analog may also be designed to becomplementary or exactly complementary to any target nucleic acidsequence of the target polynucleotide. Alternatively, the nucleic acidanalog may be designed to have a one base mismatch or a two basemismatch. In certain embodiments, the sequence of the nucleic acidanalog may be based on the sequence of PCR primers used to detectpolynucleotides associated with pathogens, the presence of a pathogen ina host, a disease gene, a genetic condition, or a genetic changeassociated with a physiological change or condition. The nucleic acidanalog may also be complementary or exactly complementary to all or partof the sequence encoding the active or functional domains of a proteinand/or the intact protein and or non-coding sequences (e.g., regulatorysequences, introns etc).

One of skill in the art, guided by this disclosure, will recognize thatin addition to nucleic acid analogs specifically listed herein, othernucleic acid analogs (including nucleic acid analogs discovered ordeveloped in the future) may be used in the methods of invention.Nucleic acid analogs that form a P/TP hybrid under the assay conditionsdescribed herein are suitable for the present methods, and affect therate of change in an optical property.

Exemplary nucleic acid sequences that can be used in the methodsdisclosed herein include but are not limited to those listed in Table 1.These and other sequences can be used with respect to targetpolynucleotides or nucleic acid analogs, and can be modified to includenon-nucleic acids located at either terminus of the nucleic acid analogor anyplace in between the termini. Useful modifications include theaddition of any natural or non-natural amino acid residues or a protein;amino acids may be present as single residues or present as apolypeptide; preferred amino acid residues include lysine and glycine.Preferred proteins that can be attached to such nucleic acid sequencesinclude those that specifically bind a ligand; more preferred proteinsinclude an antibody (specific for an antigen) and a ligand (specific fora sugar); a yet more preferred protein is biotin (specific for avidin orstrepavidin). Other sequences include complementary sequences of thoselisted in Table 1.

TABLE 1 Target Sequence (5′ to 3′) SEQ ID NO: 35S CMV promotorGATAGTGGGATTGTGCGT 1 Maize zein control ACAGTTGCTGCA 2 Maize invertaseTGTATCACAAGG 3 Maize adh CTCCGAGACCCT 4 Soy lectin CTATTGTGACCT 5Shiga-like toxin 1 TCGTTGACTACT 6 Shiga-like toxin 2 AACTGCTCCTGT 7

Nucleic acid analogs can hybridize rapidly to target polynucleotides.PNA hybridization to polynucleotides, for example, is independent ofsalt concentration. See Demidov et al., BIOCHEM. PHARMACOL. 48:1310-3(1994). PNAs are resistant to nuclease and protease attack, and bind topolynucleotides more specifically than conventional DNA probes. Shortprobes can be used with great sequence specificity (Ray and Norden,FASEB J. 14:1041-60 (2000)). Furthermore, PNA/polynucleotide hybridshave higher thermal stability than the corresponding DNA/polynucleotidehybrids, and the melting point of PNA/polynucleotide hybrids isrelatively insensitive to ionic strength, showing equal thermalstability under low (<10 mM NaCl) and moderate (500 mM NaCl) saltconcentrations. This ability of PNA/polynucleotide hybrids to form underlow salt conditions is significant because the internal structure ofdsRNA and rRNA is significantly destabilized at salt concentrationsbelow 200 mM. Therefore, assay conditions can be chosen that favor thedisruption of the target nucleic acid while still promoting formation ofPNA: DNA hybrid molecules (Stefano and Hyldig-Nielsen, DiagnosticApplications of PNA Oligomers, in DIAGNOSTIC GENE DETECTION ANDQUANTIFICATION TECHNOLOGIES19-39 (Minden ed., 1997). PNA/polynucleotidehybridization is severely affected by base mismatches and PNA moleculescan maintain sequence discrimination up to the level of a singlemismatch.

ncPNA molecules may be purchased, for example, from Panagene (Korea), orsynthesized by methods known in the art. In certain embodiments, thenucleic acid analogs can be a chiral PNA. ncPNA molecules can bysynthesized, for example, according to the methods described in Mayfieldand Corey ANAL BIOCHEM. 268(2):401-4 (1999), or Braasch et al, CURRENTPROTOCOLS IN NUCLEIC ACID CHEMISTRY. Unit 4.11 Synthesis andPurification of Peptide Nucleic Acids, pp. 14.11.11-14.11.18. John Wiley& Sons, New York. Chiral PNAs can be synthesized, for example, accordingto the methods disclosed by Kumar et al., ORG LETT. 3(9): 1269-72 (2001)or D'Costa et al., ORG LETT. 1(10): 1513-6 (1999).

In other embodiments, the nucleic acid analog includes one or more LNAs.LNAs may be purchased, for example, from Proligo (Boulder, Colo.). Instill other embodiments, the nucleic acid analog is preferably amorpholino nucleic acid analog or a TNA. TNAs can be synthesized, forexample, according to the methods disclosed by Chaput and Szostak, J.AM. CHEM. SOC. 125(31):9274-5 (2003). Morpholino nucleic acids can bepurchased, for example, from Gene Tools (Philomath, Oreg.). A comparisonbetween chiral PNA, LNA, morpholino nucleic acid analogs and non-chiralPNA analogs is disclosed in Example 3. The nucleic acid analogs producedreduced fluorescence intensities of the dye at various levels whencompared to the negative control reaction.

C. Target Polynucleotides

The target polynucleotide may be any polynucleotide, including naturallyoccurring, synthetic, and amplified polynucleotides. Other types ofpolynucleotides may be single-stranded, double-stranded,triple-stranded, or yet greater degree multi-stranded. Non-limitingexamples of target polynucleotides include DNA, RNA, regulatory RNA,mRNA, regulatory microRNA, siRNA, artificial RNA, chimeric RNA, andarmored RNA. Other non-limiting examples of target polynucleotidesinclude epigenomic DNA, epigenetic DNA, in vitro amplified DNA, andchimeric DNA. The target polynucleotide may contain single nucleotidepolymorphisms (SNPs) that are identified or quantitated by the methodsdisclosed herein. The target polynucleotide can be a nucleic acid analogalso.

D. Detergents

Any detergent can be usefully included with the reagents individually orwith the reaction mixture. Indeed, the reaction mixture preferablyincludes detergent. The advantages of adding detergent to the reactionmixture were observed and are reported in Example 1 hereof. As can beseen in detail, the addition of detergent results in greater relativesignal for test reaction mixtures as compared to negative controlreaction mixtures. Furthermore, the addition of detergent reducesphotobleaching of samples containing only dye and/or only dye andnucleic acid analog.

The detergent used in the context of the present invention, then, can bea cationic detergent, anionic detergent, nonionic detergent, orzwitterionic detergent. Non-limiting examples of cationic detergentsinclude, for example, tetramethyl ammonium chloride (TMAC) (Sigma, St.Louis Mo.). Non-limiting examples of anionic detergents includeN-lauroyl sarcosine sodium salt (LSS) and sodium dodecyl sulfate (SDS)(Sigma-Aldrich, St. Louis Mo.). Non-limiting examples of nonionicdetergents include Tween® 20, Tween® 40, Tween® 80, NP40 (Tergitol®),Triton® X-100, Span® 20, and Span® 80 (Sigma, St. Louis Mo.).Non-limiting examples of zwitterionic detergents include3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)(Sigma, St. Louis Mo.). The detergent is preferably included in themixture itself and/or in the composition that includes the dye. Suitableconcentration for the detergent ranges from about 0.01% to about 2%;more preferably from about 0.05% to about 1%; yet more preferably, about0.05%, about 0.01%, about 0.5%, or about 1%.

E. Additional Additives

Other compounds that can be added to the reaction mixture include, butare not limited to, 1,4-diazabicyclo[2,2,2]octane (DABCO),p-phenylenediamine (PPD), n-propyl gallate (NPG), ascorbic acid, sodiumazide, polyvinyl pyrrolidone (PVP), pwg-800, cyclodextran, and glycerol.

The reaction mixture can also incorporate other compounds and reagents.Examples of other compounds or reagents include Good's buffers,phosphate buffers, water, and alcohol. Suitable alcohols includebutanol, methanol, and isopropanol. Alcohol is preferably included in arange of concentration of about 1% to about 15% on a v/v basis; morepreferably, about 3%, about 5%, about 10%, or about 14% alcohol. DMSOcan also be included, at concentrations of about 1% to about 12%; morepreferably at about 10%.

F. Buffer Systems

Suitable buffers for the reaction mixture include: 50 mM KCl, 10 mM TrisHCl, and 0.1% Triton® X-100: blood lysis buffer (0.15 M NH₄Cl, 10 mMNaHCO₃, 0.1 mM EDTA pH 7.4); sucrose lysis buffer (0.32 M sucrose, 10 mMTris, 1% Triton® X-100, 5 mM MgCl₂), 1×TE pH 7.0, 7.2, 7.4, 7.6, 7.8,8.0, 8.5, 9.0, etc, phosphate-citrate buffers of 0.2M Na₂HPO₄ (fromTeknova, catalogue # S0215) and 0.1M citric acid (from Teknova,catalogue # C2440) to a final pH of 4.0, 4.2, 4.4, 4.6 and 4.8, and 10mM or 20 mM homopipes buffers pH of 4.0, 4.2, 4.4, 4.6, 4.8 and 5.0. Anyof these buffers may also include 0.05% Tween® 80.

Other useful reaction solutions can include 0.5% Tween® 20 in 5 mMphosphate, adjusted to pH 5.5; 0.5% Tween® 40 with 5 mM phosphate,adjusted to pH 5.5; 0.5% NP40 with 5 mM phosphate, adjusted to pH 5.5;0.05% lauryl sarcosine with 5 mM phosphate, adjusted to pH 5.5; or 5 mMphosphate, adjusted to pH 5.5, with 0.05% Tween® 80 and 14% methanol.

Various concentrations, pH, and combinations of the aforementionedcompounds and reagents as well, but not limited to the above, can beusefully employed with the present invention. Target polynucleotides canbe detected in samples that contain cells or tissues. The addition of adetergent preferably permeabilizes and/or lyses cells without requiringpurification or separation of polynucleotides from other components of asample. The target polynucleotide is preferably detected directly in thepermeabilized cells or crude cell lysate; the target polynucleotide doesnot need to be additionally purified or isolated from the mixture.

The addition of alcohol to a reaction mixture containing the detergentfurther reduces the photobleaching of the dye in the absence of anNAA/NA hybrid, but does not reduce the change in absorbance orfluorescence of the reaction mixture when the NAA/NA hybrid is present.This aspect of the present invention is surprising because in theabsence of detergent, the addition of alcohol slows the change inoptical property. In certain embodiments, about 8-12% ethanol or about12-15% methanol is added in tandem with a detergent.

An example showing the detection of a target polynucleotide from adetergent-treated bacterial sample will assist in describing theusefulness of both the method and the inclusion of surfactant. Withreference to Example 2 and FIG. 3, individual buffer solutions,containing various detergents, were used to permeabilize and/or lysebacterial cells. After 10 minutes incubation, 5 μl fromdetergent-treated bacterial cells were transferred to a buffercontaining achiral PNA (a nucleic acid analog) and3,3′-diethylthiacarbocyanine iodide dye, thus forming the test reactionmixture. Fluorescence intensity (535 nm excitation, 590 nm emission) ofreactions was read at time zero. Reactions were exposed to light for oneminute and re-read. This exposure-reading cycle was repeated to 10minutes total exposure. For each detergent, the “percent change” influorescence intensity at each timepoint was calculated as per theformula: 100−[RFU_(TW)−RFU_(NC)]×100, where RFU_(TW) is the measuredfluorescence intensity of the test reaction mixture, and RFU_(NC) is themeasured fluorescence intensity of the negative control mixture (i.e.,ncPNA, no target nucleic acid, dye, in buffer containing each type ofdetergent). Magnitude of percent changes corresponds to relative signalintensity (test reaction relative to negative control reaction mixtures)in the detection of the presence of the target nucleic acid sequence. Aconsistent positive percent change indicates a consistently strongersignal from the test reaction mixture as compared to the negativecontrol mixture, indicating a consistent ability for the system todetect TP. Detergents increase the relative signal, allowing the easierdetection of TP in a detergent-treated sample. The effect of somedetergents is more dramatic than that of others. Notably, the presenceof a target nucleic acid sequence can be determined from thedetergent-treated cells, without requiring further purification of thetarget polynucleotide.

The presence or quantity of a target nucleic acid in the targetpolynucleotide may be detected in any group of cells or tissues. Forexample, the presence of a target nucleic acid can be detected in atissue culture, cells, tissues or fluids obtained from an animal orplant. In other embodiments, the presence or absence of a targetpolynucleotide in any tissue or fluid can be determined. Non-limitingexamples of such tissues or fluid include urine, blood, saliva, lacrimalfluid, inflammatory exudates, synovial fluid, abscess, emphysema orother infected fluid, cerebrospinal fluid, sweat, pulmonary secretions(sputum), seminal fluid, feces, and plant tissue. Cancer cells or othercells circulating in blood can be detected, or cells having differentRNA expression levels. Other examples include detection of cell (animalor bacterial) transformation, or the absence of contamination in tissuecultures.

G. Light Stimulus

Light stimulus can be provided to a sample, nucleic acid analog, and dyemixture either concurrently with the production of the mixture or at aspecified time after the production of the mixture. The light stimuluscauses a different rate of change in an optical property of the mixture.The light stimulus may be in the visible spectrum or outside the visiblespectrum. The light stimulus may be white light of a number ofwavelengths. Alternatively, the light stimulus may be a specificwavelength or wavelengths, or range of wavelengths.

Light sources are known in the art. Different light sources result indifferent reaction rates because of differences in intensity orwavelength of the light sources. Examples of light sources include Xenonarc lamp (Ushio, #UXL-451-O), Sylvania dulux S9W CF9DS/blue and SylvaniaCool White T8-CW (OSRAM SYLVANIA, Danvers, Mass.), General ElectricT8-C50 GE Lighting, Cleveland, Ohio), Osram F9TT/50K (OSRAM GmbH,Munich, Germany), and Fritz Aurora 50/50 (Fritz Industries, Inc.,Mesquite, Tex.). Other light sources include light emitting diodes(LEDs) that produce a specific range of wavelengths, such as Jameco#183222 a 470 nm LED, Jameco #334473 a 505 nm LED, Jameco #183214 a 515nm LED, or a white multiwavelength (420-700 nm) LED #LLW5210200. LEDsemit light at least one peak wavelength, and in certain embodiments canemit light at multiple peaks. In certain variations, the bandwidth ofthe LED can be as small as 1 nm, or as large as 20 nm. Other lightsources include commercially available halogen light sources, such ashalogen headlamps (NAPA Auto Parts, Atlanta, Ga.).

The light stimulus may also have a specific intensity. In certainvariations, a 15-Watt light source at 555 nm produces between about 400foot-candles and 2000 foot-candles of illumination. In other variations,the light stimulus is one or more LEDs, preferably it is a bank of LEDs,the power of which varies from 500 μW to 4000 μW/cm² at 3.5 inches awayfrom the light.

For example, when performing the methods described herein, the lightreactive complex may be exposed to light for a period of less than 2hours. Alternatively, the light reactive complex may be exposed to lightfor a period of less than 1 hour. In yet other instances, the lightreactive complex may be exposed to light for a period of less than 30minutes. In further embodiments, the light reactive complex may beexposed to light for a period of less than 5 minutes. Other time periodsare also contemplated.

Those of skill in the art will recognize that the optimal light stimulusmay be determined without undue experimentation for a specific dye, or aspecific nucleic acid analog, polynucleotide, and dye mixture. A singleset of temperature and concentration conditions can be optimized for aspecific mixture. A source of the light stimulus can also be optimizedfor illuminating a plurality of hybridizations.

H. Forming Alternative Target Polynucleotide/Nucleic Acid Analog Hybrids

Assays for detection of target polynucleotides can be carried out usinga variety of hybridization, or association or complex formation schemes.In one format, the polynucleotide sequence may be identified byhybridization of a target polynucleotide directly to a nucleic acidanalog to form a target polynucleotide/nucleic acid analog hybrid, whichis described above. Here, we will present two additional schemes fordetecting the target polynucleotide by means of a change in the opticalproperty of the reaction mixture, as follows:

In one aspect, and as depicted in FIG. 6A, a nucleic acid analog 2molecule hybridizes to a portion of a complementary target nucleic acidtarget sequence. The nucleic acid analog can then hybridize to a secondnucleic acid analog 1 molecule that is preferably immobilized on a solidsubstrate, as depicted in FIG. 6A. This may be accomplished in aone-step or multistep process. In a one step process, the targetpolynucleotide and nucleic acid analogs are combined simultaneously. Ina multistep process, the target polynucleotide and nucleic acid analogsare combined sequentially.

In another aspect, the presence of a target polynucleotide may bedetected by forming a branched reaction crucifix form structure, anexample of which is depicted in FIG. 6B. In this format, a targetpolynucleotide is hybridized to two intermediary polynucleotides 1 and 2that are complementary to non-overlapping and contiguous portions of thetarget polynucleotide. The intermediary polynucleotides form a branchedstructure that also hybridizes to a primary nucleic acid analog. Thetarget hybridizing regions of the intermediary polynucleotides may bedesigned to be too short for a dye to facilitate the color changereaction described herein with respect to the primary nucleic acidanalog, but large enough to facilitate the color change reaction withrespect to the primary nucleic acid analogs or target polynucleotideswhen hybridized/associated.

Without relying on any particular mechanism of action, the change ofcolor or fluorescence of a dye has been found to depend on the length ofthe nucleic acid analog. The optical property contributed by the dyechanges if the nucleic acid analog is at least about 10 bases long.Accordingly, by employing a sufficient short primary nucleic acid analogin the mixture, the rate of optical change of the reaction mixture maythen be determined after the step of allowing hybridization orassociation or complex formation to occur. In the absence of thehybridization of the primary nucleic acid analog to the secondarypolynucleotides, no substantial change in the optical property of thereaction mixture is observed. Unless the intermediary polynucleotideshydridize to the target polynucleotide and to the primary nucleic acidanalog, the optical property of the reaction mixture remainssubstantially stable.

In one embodiment, the single nucleic acid analog is a universal nucleicacid analog that is used for all assays and optimized for effectivechanges in the optical property of a dye. The universal nucleic acidanalog could be used for any target nucleic acid and the secondarysequences could be varied to be specific for a given targetpolynucleotide. This scheme can be adapted to a format using animmobilized nucleic acid analog. This universal sequence can beoptimized for a given set of reaction conditions.

In another format, multiple nucleic acid analogs or targetpolynucleotides form a P/TP hybrid with adjacent regions of a targetpolynucleotide. In this format, each nucleic acid analog is preferablytoo short for the reaction mixture's optical property to change,although it does change at the background rate; but multiple nucleicacid analogs that are complementary to contiguous segments of the targetpolynucleotide preferably provide a large enough region for a rate ofchange in the optical property to result. As depicted in FIG. 6C, atarget polynucleotide as a single molecule may form a P/TP hybrid withtwo or more separate nucleic acid analogs that hybridize to adjacentsequences. SNPs can be identified using the two separate nucleic acidmolecules when there is a single base mismatch between the targetnucleic acid and one of the SNPs. If all the bases in one nucleic acidanalog cannot hybridize as in FIG. 6D then a change in optical propertymay not be observed.

I. Hybridization Conditions

The methods disclosed herein detect SNPs at room temperature. As aresult, stringent hybridization conditions do not need to be used.Although hybridization conditions can be modified or optimized,generally the hybridization conditions within a reasonable range do notaffect the ability to detect a target polynucleotide according to themethods disclosed herein.

If hybridization controls are changed or optimized, the design and/orchoice of hybridization conditions is governed by several parameters.These parameters include, but are not limited to, the degree ofcomplementarity of the nucleic acid analog to the target polynucleotide,the length of the nucleic acid analog to be utilized and the targetpolynucleotide itself. Preferred hybridization conditions allow for oneor more of the following: efficient hybridization of nucleic acidanalogs to target polynucleotides, minimization of RNA or DNA secondarystructure, minimization of RNA degradation and either discrimination ofone or more base pair changes or inclusion of one or more base pairchanges.

Hybridization reactions can be performed under conditions of different“stringency.” Conditions that effect stringency of a hybridizationreaction are widely known and published in the art. See, e.g., Sambrooket al. (2000), supra. Examples of relevant conditions include but arenot limited to, salt concentrations, pH (buffers), and temperature.Hybridization conditions utilizing lower salt concentrations generallyenhance DNA instability and PNA/polynucleotide stability. Examples ofbuffers that may be used include, but are not limited to, Na₃PO₄,NaHSO₄, K₂HPO₄, K₂SO₄, or CaSO₄. By way of example, the molarity of thebuffers may range between about 10 mM and about 0.5 M and have a pHbetween about 4 to about 10, or between about 7 to about 10, such asabout 7.0 or about 7.5. By way of example, Na₃PO₄ may be used at betweenabout 0.5 mM and about 0.5 M, such as for example, 2.5 mM, and at a pHbetween about 4 to about 10 or between about 7 to about 10, such asabout 7 or about 7.5.

Other buffer conditions include a 5 mM phosphate buffer, pH, 5.5 with0.05% NP-40 (Tergitol®). Alternatively, the 0.05% NP-40 (Tergitol®) canbe substituted with 0.05% Tween 80, optionally including about 10-14%methanol or about 8-12% ethanol. Examples of sample conditions includebut are not limited to (in order of increasing stringency): incubationtemperatures of about 25° C., about 37° C., about 50° C. and about 68°C.; buffer concentrations of 10×SSC, 6×SSC, 4×SSC, 1×SSC, 0.1×SSC (whereSSC is 0.15 M NaCl and 15 mM of any buffer as described herein) andtheir equivalents using other buffer systems; formamide concentrationsof 0%, 25%, 50%, and 75%; incubation times from about 5 minutes to about24 hours; one, two, or more washing steps; wash incubation times of one,two, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, ordeionized water. In one embodiment, hybridization and wash conditionsare done at high stringency. By way of example, hybridization may beperformed at high stringency using 50% formamide and 4×SSC followed bywashes of 2×SSC/formamide at 50° C. and with 1×SSC.

Buffers may contain ions or other compounds, or different bufferingcapacity. Alternatively a component in the buffer may have astabilization capacity; such as neomycin or other aminoglycosides, thatstabilizes triplex DNA, (Arya et al; 2003) or naphthalene diamides thatenhance triplex stability (Gianolio and McLaughlin, BIOORG. MED. CHEM.9:2329-34 (2001)), or naphthylquinoline cyanogen (Keppler et al., FEBSLETT. 447:223-6 (1999)).

J. Mutation Detection

In one embodiment, nucleic acid analogs can be used to distinguishbetween polynucleotides having an exactly complementary sequence and onewith a single base mismatch. For example and without limitation, nucleicacid analogs for use in the inventive diagnostic method may be designedto detect single nucleotide polymorphisms (SNPs). Formation of the P/TPhybrid is affected by base mismatches. According to the methods of thepresent invention, upon the addition of a dye, a single base mismatchbetween a target sequence (e.g., SNP) and a nucleic acid analog resultsin a slower rate of change in optical property of the mixture comparedto a nucleic acid analog that does not have the mismatch. Example 7depicts detection of a series of mutant SNPs. Single base mismatchesalong a double-stranded DNA were detected. FIG. 8 depicts the detectionof SNPs DNA molecules with single base-pair changes. FIG. 9 depictsdetection of DNA molecules with a two or a four base-pair change. Theidentification of SNPs for diagnosis and other methods is well known inthe art.

In another embodiment, the nucleic acid analog may be designed to detectthe presence or amount of a class of organisms. By class of organisms,it is meant that all organisms have one or more sequences that arecomplementary to, or exactly complementary to, a nucleic acid analogsequence. Such classes of organisms can be distinguished from otherorganisms based on the complementarity to nucleic acid sequences.

In yet another embodiment, the nucleic acid analog has a purine contentof less than about 60%, and has a maximum of 4 purine bases or threeguanine bases in a row. Purine-rich nucleic acid analogs tend toaggregate and have low solubility in aqueous solutions. The nucleic acidanalogs are preferably selected to minimize or avoid self-complementarysequences with inverse repeats, hairpins and palindromes because thesetypes of structures are prone to aggregate.

Nucleic acid analogs may hybridize to target polynucleotides in eitherorientation, but an anti-parallel orientation is preferred.Anti-parallel is the preferred configuration for antisense and DNAhybridization-type applications. When the orientation of the nucleicacid analog is anti-parallel, the N-terminal of the nucleic acid analogis equivalent to the 5′-end of the DNA. Both N′ and 5′ are used herein.

K. Automated Devices

Certain automated devices that can activate the reactions, record theresults and/or include the software to assist in interpretation of theresults are also contemplated within this invention. The device ispreferably fully automated and perform all reaction steps after sampleaddition. The device can be a handheld unit, a mobile unit or astationary lab unit.

In certain aspects, the device preferably detects changes in absorbance.Fluorescence detection devices can have a light source for activatingthe dye and a light source for exciting the dye. The device determinesthe emission at a range of wavelengths. Alternatively, if the devicedetects absorbance, a first light source can activate the dye and asecond light source can be used for absorbance measurements;alternatively, the instrument can use a single light source coupled withappropriate light filters. Possible test formats include but are notlimited to, a test strip (e.g., nitrocellulose or nylon), beads (e.g.,latex or polystyrene, 1-5 micron microparticles, ⅛″ or ¼″ molded orground beads), capillary tubes, monofilament, plastic test tubes, or inmicro wells on an etched surface or plastic form.

Once a sample is exposed to an activating light source and read, thedata can be gathered and stored in the device. The device preferablyautomatically tracks and calculates the change in fluorescence andproduces a read out indicating whether a sample does or does not containthe target polynucleotide, and/or determine the amount of the targetpolynucleotide. The testing format can, for example, be bar coded toprovide the unit with predetermined information on the test that may beused to increase the accuracy of the calculations.

Changes in the concentration of the dye can also be determined bymeasuring the amount of light transmitted through the reaction cellduring the reaction using a solid state photodiode sensor. This sensorproduces a current proportional to the amount of light incident on it.This photocurrent can be converted to a measurable voltage with anoperational amplifier configured to convert current to voltage. Thisvoltage can be recorded as a function of time in order to monitorchanges in dye concentration. Results can be expressed as voltage vs.time or converted to absorbance with the following formula,A _(t)=−log(V ₀ /V _(t))Where A_(t) is the absorbance at any time in the reaction, V₀ is thevoltage produced by the photo detector when no dye is present in thecell, V_(t) is the voltage produced by the photodetector at time tduring the reaction. If the dye concentration is properly chosen,typically 9-18 uM for dye I, Beers Law is obeyed and absorbance islinearly related to concentration of absorbing species by the followingequation,A=ebcwhere A is absorbance, e is molar absorptivity (M⁻¹ cm⁻¹), b is the cellpathlength in cm, and c in the molar concentration (M) of the absorbingspecies. Absorbance changes vs. time can then be measured for samplesand standards and the presence or absence of absorbing species can beinferred.

IV. METHOD FORMATS

The methods can be adapted to several different formats. Preferredformats include, without limitation, liquid-based formats,solid-substrate-based formats, and gel-based formats. It will beunderstood that various aspects of the formats can be combined.

A. Liquid-Based Formats

In one embodiment, the method for detecting a target polynucleotide isliquid-based. The sample containing the target polynucleotide, a nucleicacid analog that forms a hybrid, complex or association with a targetnucleic acid sequence of the polynucleotide in a sequence specificmanner, and the dye are preferably combined to produce a mixture inliquid solution. The mixture preferably has an optical property that canchange over time. The rate of change in the optical property of themixture is compared to a reference value characteristic of the rate ofchange in the optical property of a similar mixture containing a knownamount of a P/TP hybrid and dye to determine a relative rate of changein the optical property. The relative rate of change in the opticalproperty of the mixture correlates to the presence or amount of thespecified polynucleotide in a sample, thereby determining the presenceor amount of polynucleotide in the sample. The change in the opticalproperty of the mixture containing a nucleic acid analog or, upon addinga further step for introducing a light stimulus, a decrease in intensityof an optical property can be observed in a liquid-based format or in agel-based format or solid support format. The various methods disclosedherein may be performed in a liquid-based format.

The methods may also be conducted in any vessel, such as microfugetubes, test tubes, and chips that hold a liquid by surface tension. Themethods may also be conducted in multiwell plates or multiwell strips.The plates may contain any number of wells. In one format, 96-wellplates are used. In another format, 384-well plates are used. Any typeof plate having any number of wells can be used. The wells themselvesmay be subdivided into smaller wells. When the assay format is in amicrowell format, the liquid is retained in each well of a microtiterplate. In the alternative, or in addition, a gel matrix could be addedto one or more wells.

B. Solid Substrate-Immobilized Formats

The nucleic acid analog or target polynucleotide may be immobilized on asolid substrate. The nucleic acid analog or the target polynucleotidemay be immobilized on the solid substrate either directly or indirectlyas exemplified below.

The P/TP hybrid may be immobilized on a surface. The immobilization maybe accomplished by any suitable method known in the art, the suitabilityof which is determined functionally, i.e., that immobilization actoccurs without substantial detriment to the activity of the samecompound when free in solution. In an embodiment of this invention,either the nucleic acid analog or target polynucleotide is covalentlylinked to a second molecule that preferentially binds to a thirdmolecule on the solid substrate.

One or more components are preferably immobilized on a solid substrate.For example, either the nucleic acid analog or the target polynucleotidemay be immobilized. The P/TP hybrid may be immobilized either before orafter formation of the hybrid. The dye may be added before or afterimmobilization and/or before or after hybridization. In otherembodiments, the dye may be immobilized. Further, the light stimulus maybe added either before or after immobilization on a solid substrate.

There are many types of solid substrates that the nucleic acid analog ortarget polynucleotide molecule may be attached to, including, but notlimited to: cast membranes (e.g., nitrocellulose, nylon), ceramic,track-etched membranes (TEM), polyvinylidenedifluoride, latex,paramagnetic beads, plastic supports of all types, glass, powderedsilica or alumina on a support matrix or treated or untreated filterpaper (e.g., Whatman FTA cards). If a grid pattern is used, theimmobilized nucleic acid analog or target polynucleotide forms amicroarray. In another variation, the nucleic acid analog or targetpolynucleotide molecules are covalently modified to include a linkingmoiety, such as a biotin or amine linkage, which binds to membranescontaining the protein streptavidin or amine reactive functionality,respectively. In a further variation, the nucleic acid analog or targetpolynucleotide molecules are immobilized via sequence-specifichybridization to one or more sequences. Any means of attaching a nucleicacid analog or target polynucleotide to a support is contemplated by thepresent invention. In one aspect, the nucleic acid analog or targetpolynucleotide may be attached directly to a membrane. The nucleic acidanalog may be a PNA (e.g., Giger et al., NUCLEOTIDES AND NUCLEOSIDE17:1717 (1998)). A solution of nucleic acid analogs or targetpolynucleotides (in water) is preferably applied to a charged orchemically-modified filter and allowed to air dry. The filter is thenused for hybridization.

In another embodiment, a biotin-labeled nucleic acid analog or targetpolynucleotide is attached to a streptavidin-coated surface, such as abead or well (see, e.g., Chandler et al., ANAL. BIOCHEM. 283:241-249(2000)). Biotin-labeled nucleic acid analog or target polynucleotide canbe mixed with streptavidin-labeled latex beads. The biotin bindsstrongly with streptavidin, allowing the nucleic acid analog or targetpolynucleotide to bind to the bead in a unidirectional fashion. Thebeads are then preferably applied to a non-charged membrane with a meshsize that is 25-30% greater than the diameter of the bead. Beads becometrapped in the mesh, hence making a localized area of attached nucleicacid analogs or target polynucleotides. Direct synthesis of nucleic acidanalog or target polynucleotides on a solid substrate, such as apolypropylene membrane, may be accomplished using standard9-fluorenylmethoxycarbonyl (Fmoc) protein synthesis chemistry (see,e.g., S. Matysiak et al., BIOTECHNIQUES 31:896-904 (2001)).

In another embodiment, the nucleic acid analogs or targetpolynucleotides are fixed to a glass or other solid substrate byapplying a solution containing nucleic acid analogs or targetpolynucleotides in water directly to the glass or other support andletting it air dry.

In yet another embodiment, a nucleic acid analog is designed to producea net positive charge, and may bind a negatively charged membrane. Forexample, a positively-charged lysine or glycine at a 5′ or 3′ end of thenucleic acid analog molecule may be used to attach the nucleic acidanalog molecule to a negatively-changed nylon membrane. The negativelycharged membrane repels any nucleic acid that is not complementaryand/or exactly complementary to the nucleic acid analog, thus minimizingnon-specific binding.

In yet another embodiment, nucleic acid analogs or targetpolynucleotides are preferably attached to microspheres as described byXu et al., NUC. ACIDS RES. 31:e43 (2003). For each conjugation reaction,approximately 4×10⁶ carboxylated microspheres (Polysciences, Warrington,Pa.) can be pelleted and washed with 0.1 M imidazole buffer pH 7.0.After resuspension of microspheres in 20 μl of imidazole buffer pH 7.0(10% solids), 1 μl of 100 mM oligos, and 100 μl of 200 mM EDAC (Acros,Pittsburgh, Pa.) in freshly made imidazole buffer pH 7.0 is added andthe reaction mixture is incubated for about 2 hours at room temperaturewith continuous rotation. An additional 100 μl of 200 mM EDAC in freshlymade imidazole buffer pH 7.0 is added and the room temperatureincubation with rotation was continued for another 2 hours. Microsphereswere then centrifuged, washed twice with water, and resuspended in 40 μlof phosphate-buffered saline (PBS), pH 7.4.

In other embodiments, nucleic acid analogs or target polynucleotides areattached to microspheres, as described by Running and Urdea,BIOTECHNIQUES 8:276 (1990).

Any target polynucleotide, or group of target polynucleotides, may bedetected using the solid substrate-based system. In this case, a solidsubstrate contains multiple nucleic acid analogs or targetpolynucleotides immobilized on a solid substrate. A negative controlnucleic acid analog or target polynucleotide that does not form nucleicacid analog/polynucleotide hybrid is preferably included on the solidsubstrate. A positive control nucleic acid analog that always forms anucleic acid analog/polynucleotide hybrid is preferably included on thesolid substrate.

An end of a monofilament can also serve as a solid substrate. In such anembodiment, a nucleic acid analog is preferably immobilized on the endof a monofilament, and a dye and target polynucleotide are preferablyadded to the tip. When light is applied to the end of the monofilament,the change in an optical property of the dye is monitored over time.

Fluorescent nanobeads such as quantum dots (Invitrogen Corporation,Carlsbad, Calif.) can serve as the solid substrate. Quantum dots coresare composed of semiconductor cadmium salts that fluoresce at differentwavelengths based on salt and size. This core is enveloped in a shellthat is composed of a non-emissive transparent, but structurallyrelated, material that can be efficiently wed to the underlying corematerial. In this way, the core molecules are tricked into sensing whatstill appears to be a virtually infinite array of atoms in everydirection; little or no reorganization is necessary and the fluorescentemission remains stable and bright over a long period of time.

In another embodiment, nucleic acid analogs are preferably conjugated toquantum dots that emit at different wavelengths. A single sequence canbe conjugated to dots of a single wavelength or one or more sequencescan be conjugated to dots of a single wavelength. These quantum dots ofeither a single or multiple wavelengths are preferably mixed with apolynucleotide mixture allowing hybrids to form. The dots with hybridsattached may be washed to remove non-complementary polynucleotide. Amixture with dye and reaction buffer is added to the dots. The dots withhybrid are then dispensed in fraction wells of a multiwell plate asdescribed at U.S. Pat. No. 6,838,243. In each well there are manyfraction wells. The fraction wells are of the correct size that each canaccommodate a single dot. The amount of liquid dispersed is such thatthe fraction wells are not quite full. The mixture is then exposed to anactivating light. After a period of time (or at multiple times) thecolors/fluorescence of the fraction wells are determined. Thefluorescence of the quantum dot identifies the target sequence and thedisappearance of an optical property in the reaction indicates thepresence of the target sequence in the polynucleotide mixture.

The reactions can be quantitative based on the number of quantum dotsbioconjugated with a given sequence indicating the presence of thatsequence. Other types of beads may be used, including but not limited toLuminex® beads (MiraiBio, Alameda, Calif.). In some embodiments, beadsmay be magnetic to assist in the washing. Centrifugation may be involvedin the washing.

In certain embodiments, a solid support with a specific background colorprovides significantly improved detection of the target polynucleotide.Specific embodiments of solid supports can have any color backgroundknown in the art. In one particular embodiment, the solid support has awhite background. The solid support can be a microtiter plate having aspecific color of well, such as a microtiter plate having a whitebackground. Alternatively, the compositions can include a sample surfacemodified to have the same interior as any plate described herein.

C. Gel-Based Formats

The methods disclosed herein can be used to determine the presence oramount of a target polynucleotide in a gel based assay. A gel, such asan agarose or acrylamide gel, that contains the dye is prepared.Alternatively, the dye is added to the gel after it is run or the dye isadded to the sample before it is loaded onto the gel. A sample that maycontain, does not contain, or is suspected of containing, or suspectedof not containing, a target polynucleotide is added to the gel. Thecomponents of the sample can be separated from each other on the gel. Anucleic acid analog that is complementary to a target nucleic acidsequence of a target polynucleotide is added either before or afterdigestion of the sample polynucleotide but before an electric current isapplied to the gel. Alternatively, the nucleic acid analog can be addedafter an electric field is applied to the gel. After the bands areallowed to migrate on the gel, a light stimulus is applied to the gel.

After the light stimulus is applied to the gel, the portion of the gelcontaining the P/TP hybrid has at least one different optical propertyrelative to the rest of the gel. In one exemplary embodiment, when thegel is combined with 3,3′-diethylthiacarbocyanine iodide dye and exposedto a light stimulus, visually the resulting gel lacks any color (apartfrom the agarose-colored portions) where the P/TP hybrid is present. Theremaining portion of the gel has a detectable color. In addition, byillumination with a 254 nm transilluminator, the gel will fluoresceexcept where the P/TP hybrid is absent. In the region of the P/TPhybrid, there is no fluorescence. The presence of a target nucleic acidsequence in a target polynucleotide is thereby identified on a gel.

The ability to determine the presence of a target polynucleotidesequence on a gel can be adapted to any number of conventional molecularbiology techniques. For example, a conventional Southern blot can beadapted to the methods of determining the presence of a targetpolynucleotide on a gel. Target polynucleotides are digested by aconventional restriction digest and run on a conventional agarose gelthat contains dye. A nucleic acid analog can be added to the targetpolynucleotides before, during, and/or after restriction digestion. Incertain formats, the target nucleic acids can be denatured. Instead oftransferring the polynucleotides to a membrane, the gel is exposed tolight stimulus. The loss of fluorescence intensity of the dyecorresponds to the location of a target polynucleotide containing thetarget nucleic acid sequence. In an alternative method, the nucleic acidanalog is added to the gel. In various non-limiting embodiments, boththe nucleic acid analog and the dye are added to the gel after thepolynucleotides have been separated by the electric current. Theelectrically separated polynucleotides can be transferred to a membraneand the nucleic acid analog allowed to hybridize to the immobilizedpolynucleotides that have the complementary sequence. This membrane isthen immersed in liquid containing dye and other reaction components,after some period of time the membrane is removed from the liquid andexposed to light stimulus. The presence of the complementarypolynucleotide would be indicated by a clear spot on the membrane.

Unlike conventional Southern blot analysis, the presently describedmethod does not require heat or salt-based denaturation of the nucleicacid, and does not require blotting to identify target polynucleotideshaving a specific nucleic acid sequence, that is the hybridization couldoccur before or after transfer of the polynucleotides or transfer is noteven required.

The methods disclosed herein can also be adapted to a northwestern blot.A “Northwestern” analysis is the generic term for studying protein-RNAinteractions in either a solution-phase format, a gel (electrophoresis)format, or a substrate (such as a membrane) blot format. A protein maybind to RNA in a sequence-specific manner (e.g., HIV tat protein bindingto the TAR sequence) or in a sequence-independent manner (e.g.,RNA-binding proteins). The methods can be used to identify specificsequences within RNA that are relevant to protein binding. For example,into a solution containing a RNA segment and a protein (thought to bindto a particular RNA sequence) is added a complementary nucleic analogand the dye. Upon exposure to light, the solution either changes colorindicating that the nucleic acid analog hybridized to the RNA and theprotein did not, or the solution does not change color indicating thatthe protein has bound to the RNA effectively blocking the nucleic acidanalog from hybridizing.

The same methods can be performed in a gel electrophoresis format wherethe gel contains the dye. The gel is stained post electrophoresis. Thesample containing the RNA, the protein, and the nucleic acid analogcould be electrophoresed in a gel matrix followed by exposure of the gelto light. Identification of the band corresponding to nucleic acidanalog hybridizing to the RNA is visualized by a loss of color (or“hole”) in the gel. Absence of the “hole” would be indicative ofprotein-RNA interaction in a sequence-specific manner, therebyinhibiting binding of the NAA.

Similarly, for substrate blot experiments, the protein (or RNA) could beimmobilized to a substrate, followed by incubations with RNA sequences(or protein), followed by incubations with the nucleic acid analog anddye, followed by exposure to light. The loss of color would indicatehybridization of the NAA at a specific RNA sequence not bound to theprotein. The presence of color would indicate a protein —RNA interactionat the specific sequence inhibiting binding of the NAA.

Modification of a nucleic acid analog with a positively-charged moleculecan be used to increase specificity of hybridization. For example, anucleic acid analog can be modified to contain a positive charge orlinked to a molecule or molecules having a positive charge. The positivecurrent in a gel directs the positively charged nucleic acid analog andthe target polynucleotide in opposite directions. The greater the numberof hydrogen bonds between the nucleic acid analog bases and the targetpolynucleotide bases, the greater the likelihood that the P/TP hybridwill remain annealed. Depending on the current and potential appliedacross the gel, P/TP hybrids containing mismatched sequences can bepulled apart, resulting in denaturation of the P/TP hybrid. The absenceof a hybrid will result in a baseline change in an optical propertywithin/of the gel.

The methods of determining the presence or quantity of a targetpolynucleotide on a gel can be adapted to any gel-based method. Forexample, determining the presence of a target polynucleotide on a gelcan be adapted to conventional Northern blot analysis, in which thetarget polynucleotide is RNA, not DNA. The methods of determining thepresence or quantity of a target polynucleotide on a gel also can beadapted to northwestern analysis. Conventional methods are furtherdisclosed, for example, in Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook et al., 2000) Cold Spring Harbor Press and MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989)Cold Spring Harbor Press, both of which are incorporated herein byreference in their entirety.

D. Detection of Polynucleotides Involved in Genetic Manipulation.

The different rate of change of an optical property of a mixture (orgel) in the presence of an P/TP hybrid can be used to screen fortransformation of bacterial colonies in a colony dot-blot. Inconventional dot-blot assays, putative transformed cells are grown intocolonies on medium. The colonies are transferred to a membrane, theirlocation is fixed, the cells are lysed, and the polynucleotides areattached to the membrane.

The dot blot can be adapted using the methods disclosed herein todetermine the presence of target polynucleotide in transformed phage,other viral particles, genetic material, or detection of transfection orinfection of eukaryotic cells. The detection of the targetpolynucleotide attached to the membrane could occur via several methods.In one non-limiting example, a membrane is washed and a nucleic acidanalog that is complementary to the sequence of interest or designed toshow disruption of the sequence of interest. After a wash step (s) themembrane is placed on a gel-based film that contains the dye. Thissandwich is exposed to a light stimulus. Areas in which color is reducedor disappears (or fluorescent emission is reduced or disappears)indicate the presence of the P/TP hybrid and thus the polynucleotidesequence of interest.

In another variation, the nucleic acid analog and the dye may be both inthe stationary phase of the gel and simultaneously sandwiched to themembrane with the attached polynucleotides. In yet another variation,the dye (with or without the nucleic acid analog) can be in a liquid gelthat is poured on the membrane and areas in which color or fluorescentemission is reduced or gone after exposure to light stimulus indicatethe presence (and location) of colonies with the sequence of interest.In a further variation, the colonies may not be transferred to amembrane, but are preferably rather lysed on the plate and any of theabove detection schemes applied. In a yet further variation, thepolynucleotides may never be attached to the membrane and allowed tointeract with reaction components held in a stationary phase (such as agel).

Alternatively the colonies may be picked into a reaction vessel and thereactions with the change in an optical property of a dye occur in thereaction vessel.

V. TARGET MOLECULE CAPTURE ASSAYS A. Capture

In one aspect, the present invention relates to novel methods forisolating a target molecule from a sample suspected of containing thetarget molecule. As described in more detail below, the methods of thepresent invention may be employed to isolate any type of molecule forwhich there exists a binding molecule capable of binding specifically ornon-specifically to the target molecule and which can be linked,directly or indirectly, to a nucleic acid molecule. Target molecules mayinclude, for example, polynucleotide molecules, as well asnon-polynucleotide molecules, such as protein molecules, polypeptidemolecules, peptide molecules, oligosaccharide molecules, and otherchemical substances, to name only a few.

In accordance with the present invention, it has been discovered thatthe combination of

-   -   (i) a polynucleotide linked to target molecule,    -   (ii) a nucleic acid analog that is complementary to the        polynucleotide and is bound, directly or indirectly, to a solid        substrate, and    -   (iii) a light reactive dye,    -   associate to form a light reactive complex bound to the solid        substrate, which can then be separated as a unit (the solid        substrate together with the complex), based on a property of the        solid substrate or of the complex as whole, to thereby isolate        the target polynucleotide.

The light reactive complexes of the present invention, and the methodsfor making and using such complexes, have been shown to be useful inmany applications. Advantageously, the methods of the invention,involving capture of the target molecule and, optionally, release underlight exposure, can be accomplished within relative brief periods oftime, and under ambient light and temperature conditions, allowing themethod to be performed without equipment typically required for nucleicacid hybridization and melting conditions used in polymerase chainreaction or other hybridization methods, while still achieving anacceptable level of specificity and sensitivity. The methods of thepresent invention may therefore be advantageously used, for example, inthe field outside controlled laboratory conditions, for the detectionand analysis of a multiplicity of compounds, including both chemical andbiological compounds.

The mechanism of action involved in light reactive complex formation andrelease involved in any of the capture, release and detection steps ofthe present invention is not yet known. Examples illustrating themethods of capture/release using the immobilized light reactivecomplexes of the present invention are provided in Examples 29, 30 and31, below. While these examples illustrate typical conditions underwhich light reactive complexes are formed, it is understood that suchconditions may be varied and/or optimized in accordance with theparticular objectives and results desired.

In one embodiment of the present invention, the methods of the presentinvention are used to isolate a target molecule that is apolynucleotide, such as a DNA or RNA molecule, by means of a directinteraction between a target polynucleotide and a bound nucleic acidanalog polynucleotide that is complementary to the target polynucleotideor a portion thereof and is immobilized on a solid substrate. Inaccordance with such embodiments, the methods of the invention comprisethe step of combining the following components:

-   -   (i) a sample suspected of containing the target polynucleotide;    -   (ii) a first nucleic acid analog immobilized to a solid        substrate, wherein the first nucleic acid analog is        complementary to a portion of the target polynucleotide; and    -   (iii) a first light reactive dye;        to form, if the target polynucleotide is present in the sample,        a light reactive complex comprising the target polynucleotide,        the first nucleic acid analog, and the light reactive dye. In a        subsequent step, the solid substrate and the light reactive        complex immobilized thereon is separated from the sample, based        on a property of the solid substrate, to thereby isolate the        target polynucleotide from the sample.

In another embodiment, the methods of the present invention are used toisolate a target molecule that is a polynucleotide, such as a DNA or RNAmolecule, by means of an indirect interaction between a target moleculeand a capture polynucleotide immobilized on a solid substrate. In theseembodiments, various intermediary constructs are used to link the targetmolecule, which may be a polynucleotide, a polypeptide, a sugarmolecule, etc., with a bound polynucleotide immobilized to a solidsupport. The intermediary constructs are designed so as to provide atleast one pair of complementary polynucleotides, at least onepolynucleotide of which is a nucleic acid analog, such as, for example,a PNA polynucleotide, an LNA polynucleotide, or morpholinopolynucleotide, that can associate with a light reactive dye to form alight reactive complex.

In one embodiment, illustrating an indirect interaction between a targetmolecule and a capture nucleic acid analog polynucleotide immobilized ona solid substrate, the present invention relates to a method ofisolating a target molecule from a sample suspected of containing thetarget molecule, comprising the step of combining the following:

-   -   (i) a sample suspected of containing a target molecule;    -   (ii) a bound polynucleotide immobilized on a solid substrate;    -   (ii) a chimeric molecule comprising (1) a bridging        polynucleotide complementary to the bound polynucleotide,        and (2) a target component capable of binding to a portion of        the target molecule; and    -   (iv) a light reactive dye.

In this particular embodiment, the target component binds to the targetmolecule, if present, and the target component is linked to a bridgingpolynucleotide (the target component and the bridging polynucleotide arecollectively referred to as the “chimeric molecule”). The bridgingpolynucleotide is complementary to another polynucleotide that isdirectly or indirectly linked to the bound polynucleotide immobilized onthe solid substrate. At least one of the (1) the bound polynucleotideimmobilized on the solid substrate, and (2) the bridging polynucleotideof the chimeric molecule, is a nucleic acid analog polynucleotide thatforms a light reactive complex with a complementary polynucleotide andthe reactive dye which is immobilized on a solid substrate. Thus, theindirect interaction requires at least one complementary pair ofpolynucleotides that can associate with the light reactive dye to form alight reactive complex.

In some embodiments of the invention, the immobilized polynucleotide isa nucleic acid analog polynucleotide. For example, in some embodiments,the target molecule is a polynucleotide, the target component of thechimeric molecule is a nucleic acid analog polynucleotide complementaryto the polynucleotide target molecule, the bridging polynucleotide ofthe chimeric molecule is a non-nucleic acid analog molecule, and thebound polynucleotide immobilized on the solid support is a nucleic acidanalog polynucleotide that is complementary to the bridgingpolynucleotide.

In other embodiments, the immobilized polynucleotide is a non-nucleicacid analog polynucleotide and the bridging polynucleotide is a nucleicacid analog polynucleotide. For example, in some embodiments the targetmolecule is a polynucleotide, the target component of the chimericmolecule is a nucleic acid analog polynucleotide complementary to thepolynucleotide target molecule, the bridging polynucleotide of thechimeric molecule is a nucleic acid analog molecule, and the boundpolynucleotide immobilized on the solid support is a non-nucleic acidanalog polynucleotide that is complementary to the bridgingpolynucleotide.

In yet another embodiment, illustrating another indirect interactionbetween a target molecule and a capture nucleic acid analogpolynucleotide immobilized on a solid substrate, the present inventionrelates to a method of isolating a target molecule from a samplesuspected of containing the target molecule, comprising the step ofcombining the following:

-   -   (i) a sample suspected of containing a target molecule,    -   (ii) a bound polynucleotide immobilized on a solid substrate,    -   (iii) an intermediary binding polynucleotide having a first        binding region complementary to the bound polynucleotide and a        second binding region,    -   (iv) a chimeric molecule comprising (1) a bridging        polynucleotide complementary to the second binding region of the        intermediary binding polynucleotide, and (2) a target component        capable of specifically binding to the target molecule, and    -   (v) a light reactive dye.

In this embodiment, the target component binds to the target molecule,if present, and the target component is linked to a bridgingpolynucleotide. In contrast to the embodiment described in the precedingparagraph (where the bridging polynucleotide forms a complex directlywith the bound polynucleotide immobilized on the solid substrate), thisembodiment contemplates the use of an intermediary bindingpolynucleotide, having a first binding region complementary to the boundpolynucleotide immobilized on the solid substrate, and a second bindingregion that is complementary to the bridging polynucleotide of thechimeric molecule to which the target molecule is linked. In thisconstruct, there are at least two regions of complementarity betweenseparate polynucleotides: the first region of complementarity is betweenthe bound polynucleotide and the first binding region of theintermediary binding polynucleotide; the second region ofcomplementarity is between the bridging polynucleotide and the secondbinding region of the intermediary binding polynucleotide. Either one orboth of these regions may form a light reactive complex with a lightreactive dye, provided that at least one of the polynucleotides of thecomplementary pair comprise a nucleic acid analog, such as, for example,a PNA polynucleotide, an LNA polynucleotide, or a morpholinopolynucleotide, which nucleic acid analog polynucleotide can associatewith its complementary polynucleotide and a light reactive dye to form alight reactive complex (immobilized on a solid substrate). Thus, atleast one of (1) the bound polynucleotide immobilized on the solidsubstrate, (2) the first binding region of the intermediary bindingpolynucleotide, (3) the second binding region of the intermediarybinding polynucleotide, and (4) the bridging polynucleotide of thechimeric molecule, is a nucleic acid analog polynucleotide that forms alight reactive complex with a complementary polynucleotide and thereactive dye which is immobilized on a solid substrate.

In some embodiments, the bound polynucleotide immobilized on the solidsubstrate is a non-nucleic acid analog polynucleotide, the bridgingpolynucleotide of the chimeric molecule is a non-nucleic acid analogpolynucleotide, and the intermediary polynucleotide is a nucleic acidanalog polynucleotide.

In other embodiments, the bound polynucleotide immobilized on the solidsubstrate is a nucleic acid analog polynucleotide, the bridgingpolynucleotide of the chimeric molecule is a nucleic acid analogpolynucleotide, and the intermediary polynucleotide is a non-nucleicacid analog polynucleotide.

In yet other embodiments, the first portion of the intermediarypolynucleotide is a non-nucleic acid analog polynucleotide, the boundpolynucleotide immobilized on the solid substrate is a nucleic acidanalog polynucleotide, the second portion of the intermediarypolynucleotide is a nucleic acid analog polynucleotide, and the bridgingpolynucleotide of the chimeric molecule is a non-nucleic acid analogpolynucleotide.

In yet other embodiments, the first portion of the intermediarypolynucleotide is a nucleic acid analog polynucleotide, the boundpolynucleotide immobilized on the solid substrate is a non-nucleic acidanalog polynucleotide, the second portion of the intermediarypolynucleotide is a non-nucleic acid analog polynucleotide, and thebridging polynucleotide of the chimeric molecule is a nucleic acidanalog polynucleotide.

It is contemplated that the methods of the present invention may be usedto capture and isolate any number of different types of targetmolecules. For example, in some embodiments of the invention, the targetmolecule is a polynucleotide and the target component of the chimericmolecule is a nucleic acid analog polynucleotide complementary to thepolynucleotide target molecule.

In other embodiments, the immobilized polynucleotide is a nucleic acidanalog polynucleotide and the bridging polynucleotide is a non-nucleicacid analog polynucleotide.

In still other embodiments, the target molecule is a polynucleotide, thetarget component of the chimeric molecule is a nucleic acid analogpolynucleotide complementary to the polynucleotide target molecule, thebridging polynucleotide of the chimeric molecule is a non-nucleic acidanalog molecule, and the bound polynucleotide immobilized on the solidsupport is a nucleic acid analog polynucleotide that is complementary tothe bridging polynucleotide.

As will be appreciated by those skilled in the art, other combinationsof polypeptide and non-polypeptide linkages may be utilized toaccomplish the purposes of the invention, provided that at least onecomplementary polynucleotide pair comprises at least one nucleic acidanalog that can form a complex with a complementary polynucleotide and alight reactive dye.

B. Separation

The steps described in the above embodiments illustrate various means bywhich a target molecule of interest can be captured and immobilized on asolid substrate. In another aspect, the methods of the present inventioninclude the step of separating the solid substrate (to which the targetmolecule is directly or indirectly attached) from the mixture, tothereby isolate the target molecule. Solid substrates include any of thevarious substrates used for separation, purification and detection ofbiological substances, including solid beads, gels, and the like, asdiscussed above in more detail. Thus, in one embodiment of theinvention, the target molecule immobilized to the solid substrate via atleast one light reactive complex, is isolated from a mixture, based on aproperty of the solid substrate, to thereby isolate the targetpolynucleotide from the sample. It will be understood that isolation ofthe solid substrate/target polynucleotide complex from the sample mayinclude removal of the solid substrate from the sample mixture, orremoval of the sample mixture from the solid substrate by washing thesolid substrate with appropriate washing solutions, or combinations ofthe foregoing.

In typical applications, the target molecule will originate from aheterogeneous mixture, such as a blood sample, a water sample, a tissuesample, etc., comprising various contaminants mixed with the targetmolecule of interest. The methods of the present invention areparticularly useful in separating a target molecule from such aheterogeneous mixture. As described in more detail in the examplesbelow, various solid substrates may be used to immobilize a targetmolecule. Such immobilization techniques are well known in the art, asare techniques for the separation of a solid substrate from componentsnot bound to the substrate, and need not be described herein.

C. Light Conditions

As illustrated in the examples, one of the principal advantages of themethods of the invention is that complexes formed a with a specifictarget molecule can be isolated and released under ordinary ambientlight conditions. Capture/release experiments have been shown to work inordinary room conditions, with no sunlight and only the light from thefluorescent overhead light fixtures. Light conditions were observedusing a LaserCheck light meter, which indicated that the light level wasapproximately 66.7 μW/cm² in the room with fluorescent lights on andwindows, and 34.1 μW/cm² in the room with fluorescent lights on and nowindows. Other than the location of the release step, thecapture/release/detection reactions were the same. The initial slope at2 minutes was 68.5 (std. dev. 2.8) for the reactions released in the lab(with lights and windows). The initial slope at 2 minutes was 70.8 (std.dev. 0.7) for the reactions released in the room (with lights and nowindows).

In some instances, the present invention may also be practiced undercontrolled light conditions, using an instrument that carefully controlslight level, wavelength and exposure time, so as to provide more controlover assay results.

D. Temperature Conditions

In accordance with the present invention, the capture of targetmolecules by means of light reactive complex formation may beaccomplished under ambient temperature conditions. In some embodiments,the light reactive complex is formed at room temperature. In otherembodiments, the light reactive complex is formed at a temperature lessthan about 37° C. In yet other embodiments, the light reactive complexis formed at a temperature less than about 45° C. The above lightconditions are substantially below those temperature conditionsordinarily required for the rapid formation of complexes betweencomplementary polynucleotide molecules.

E. Release

In yet another aspect of the present invention, the target molecule,captured via formation of a light reactive complex between complementarypolynucleotides immobilized to a solid substrate, is released from thelight reactive complex.

In one embodiment, the present invention optionally includes the step ofexposing the light reactive complex, formed as described above, tolight, to thereby elute the target polynucleotide from the solidsubstrate into the eluant to provide a purified target polynucleotide.

Any one or more of the light reactive dyes described herein may be usedin the capture assays of the present invention. The light reactive dyescapable of being used in the methods of the invention are described inmore detail in the definition of “dye,” above, and in section IV.A,below. For example, some preferred light reactive dyes include thefollowing: 3,3′-diethylthiacarbocyanine, 3,3′-diallylthiacarbocyanine,3,3′-diethyl-9-methylthiacarbocyanine, 3,3′-dibutylthiacarbocyanine,3,3′-dipropylthiacarbocyanine, 3,3′-dipentylthiacarbocyanine, and1,1′-diethyl-2,2′-carbocyanine, and salts thereof. One dye found to beparticularly useful is 3,3′-Diethylthiacarbocyanine or salts thereof.Preferred salts include, for example, iodide and bromide salts.

The nucleic acid analogs used in the various embodiments of the captureand release assays have been described previously herein. In someembodiments of the invention, the nucleic acid analogs include, forexample, PNA polynucleotides, LNA polynucleotides and morpholinopolynucleotides. PNA polynucleotides have been found to be especiallyamenable to light reactive complex formation with the light reactivedyes of the invention. As discussed elsewhere, the nucleic acid analogsmay be of various lengths. Particularly useful nucleic acid analogsrange from 10-30 bases in length, from 12-25 bases in length, less thanabout 20 bases in length, or less than about 15 bases in length. In someembodiments, the nucleic acid analogs are about 17 bases in length. Thelength of particular nucleic acid analogs can vary considerably;however, due to the enhanced stability of the light reactive complexesof the invention (which comprise nucleic acid analog polypeptides havinga greater degree of stability, together with the light reactive dye) thecapture assays of the present invention enable use of nucleic acidanalog polynucleotides of shorter length than ordinarily used for PCRbased probes and primers, while still achieving acceptable levels ofspecificity and sensitivity.

The capture and release assays described above may be used to isolate atarget molecule from a heterogeneous mixture, preparatory to detectionin accordance with the detection methods described herein, which mayutilize light reactive complexes formed between polynucleotides andnucleic acid analog polynucleotides. It is understood that the samenucleic acid analogs used in conjunction with the target moleculecapture and release steps of the invention, may also be use in thesubsequent detection steps. Alternatively, it may be desirable in somecircumstances to utilize nucleic acid analog polynucleotides that arecomplementary to entirely different polynucleotides. For example, in thecase of nucleic acid analog polynucleotides that are complementary to,and form a complex directly with, a target polynucleotide itself, thenucleic acid analog polynucleotides used in the capture or release stepsmay be designed to be complementary to a completely different region ofthe target polynucleotide. Although the mechanism of action responsiblefor light induced dissolution of the light reactive complexes isunknown, experiments have demonstrated that under some conditions theregion of complementarity between a polypeptide and a nucleic acidanalog, which forms a complex with the light reactive dye, is notsubsequent to complex dissolution, amenable to hybridization withcomplementary nucleic acid primers, and cannot, for example, be used asa primer site for PCR amplification. Although data indicates that thecomplex is no longer associated with the solid substrate to which is waspreviously bound, this phenomena may be explained by any one of varioustheories: (1) the complex may be intact (i.e., the binding site of theprimer may be inaccessible because it is occupied by the dye, thecomplementary analog nucleic acid polypeptide, or both, and thereforeunavailable for hybridization), (2) the complex may be destroy (i.e., nocomplementary binding site exists), or (3) may be modified in such amanner that it is no longer amenable to hybridization with acomplementary polynucleotide. In any event, in those circumstances wherethe region of complementarity of a target polynucleotide cannot be usedin subsequent steps, it is possible to utilize nucleic acid analogpolynucleotides that are complementary to different regions of thetarget polynucleotide.

In view of the fact that the mechanism of action for dissolution of thelight reactive complexes from the solid substrate is not known, nor isit known whether the complex is partially or completely intact, ordestroyed, it is to be understood that references to “eluting” thetarget polynucleotide from the solid substrate into the eluant toprovide a purified target polynucleotide does not require or imply thatthe target polynucleotide itself remains intact or retains biologicalactivity. As described in the above paragraph, the target molecule maybecome disassociated from the solid substrate to which is wasimmobilized, and exist in any of a number of forms, remaining complexedwith one or more of the nucleic acid analog polynucleotide and the dye,or the binding region to which the complementary nucleic acid analogpolynucleotide may be disrupted by the light reactive dye upon exposureto light, resulting in destruction and/or cleavage of the region ofcomplementarity. Thus, the term “eluting” is intended to be construedliberally to mean that the solid substrate is disassociated, released orseparated from the target molecule, without implying the state orcondition of the target molecule or the light reactive complex (or ofthe solid support) with which is was associated prior to exposure tolight.

VI. QUANTIFYING THE AMOUNT OF A TARGET POLYNUCLEOTIDE

The methods and compositions disclosed herein may be used to quantifythe amount of target polynucleotide in a sample. In one embodiment, theamount of a target polynucleotide may be detected by establishing serialdilutions of the nucleic acid analog molecule, adding various amounts ofthe target polynucleotide samples, and comparing the samples to controlsof known concentrations. In another embodiment, the amount of a targetpolynucleotide may be detected by establishing serial dilutions of thetarget polynucleotide, adding various amounts of the nucleic acidanalogs or target polynucleotides, and comparing the samples to controlsof known concentrations.

Alternatively, the amount of a target polynucleotide can be detected bymeasuring the kinetics of the assay based on time. Measurements of thedye in the combined mixture are taken at regular intervals afterpreparation of the mixture, or after application of light stimulus. Thedye may be detected at distinct times after combination of the mixture,or after application of the light stimulus. The time may be any fixedtime, for example the total time for the change in optical property, orthe time required for the optical property to have changed by a certainpercentage, such as, but not limited to, about 20%. The reactions can befrozen (further change stopped), for example with the addition ofsolvents such as 20% methanol, 15% isopropanol, 15% DMSO, or 10%butanol.

The quantity of polynucleotide in a sample may be determined afterexposure to the light stimulus. The change in the optical property ofthe dye may be measured following pre-exposure to the light stimulus forthe starting optical property. Measurements may be taken at distincttimes (for example, but not limited to, taken at 30 second intervals, 1second intervals, millisecond intervals, or microsecond intervals) afterexposure to the light stimulus. The reactions can be frozen (furtherchange stopped) as described above.

Changes in the sample due to exposure to the light stimulus can beobserved in several ways. The change in the optical property may beobserved as a change in color, absorbance, transmittance, fluorescence,reflectance, chemiluminescence, or a combination thereof. Alternatively,the change in optical property can be read using a reader. This changeis measured using a spectrophotometer or a fluorometer, such as TecanGenios or a Tecan Safire. Specific observation wavelengths may beselected, for example by a filter. A positive control expresses a changein absorbance faster than a negative test. It can be measured as adifference in the rate of change, or the difference in the change at aset time. If a light stimulus is used and fluorescent properties areobserved, the light stimulus provided to the sample is at a higherenergy (lower wavelength) than the observed emission. The excitation maybe at, for example, 535 nm and the emission may be read at 590 nm. Thefluorescence may be measured as a difference in the rate of change orthe difference in the change at a set time or at a minimum time.

VII. TARGET BINDING COMPLEXES

The methods and associated compositions disclosed herein can also beused as a reporter to facilitate identification of molecularinteractions, such as protein-protein interactions orprotein-glycoprotein interactions.

A target binding complex includes a target binding component and areporter complex. Target binding components, discussed in more detailbelow, are any molecule that is capable of binding a target. A reportercomplex includes a first reporter nucleotide sequence, a second reporternucleotide sequence, and a dye. At least one component of the reportercomplex is covalently bonded to a target binding component to form amodified target binding component. The modified target binding componentis introduced to a sample suspected of containing a target. Preferably,washing steps are performed to remove target binding component that isnot bound to the target. The remaining components of the reportercomplex are added to form a target binding complex. The order ofaddition of the target binding complex or the remaining components ofthe reporter complex to the sample is not critical, and can be inreverse order. Optionally, a light stimulus is provided; preferably, thelight stimulus is provided. The rate of change in an optical property ofthe sample (now including the dye) is determined, as described above forP/TP hybrids. The presence or amount of the target is therebydetermined.

It will be understood that any other components described in the methodsin the present invention can be added to the target binding complex inany combination.

Further, it will be understood that methods of detecting targets can beperformed using any format disclosed herein. For example, thetarget-binding complex can be adapted to a liquid-based format,solid-based format, or gel-based format. Non-limiting examples offormats include gel matrix platforms, electrophoretic platforms,membrane-bound platforms, chromatographic platforms, immobilized plates,and immobilized beads.

A. Reporter Complex

The reporter complex is similar to, and in some cases the same as, thecombination of target polynucleotide, nucleic acid analog, and dyedisclosed in the methods of detecting a target polynucleotide. As notedabove, the reporter complex includes a first reporter nucleotidesequence, a second reporter nucleotide sequence, and a dye. The firstreporter nucleotide sequence can be a DNA, RNA, or a nucleic acidanalog. Likewise, the second reporter nucleotide sequence can be,independently of the first reporter nucleotide, a DNA, RNA, or a nucleicacid analog. The first and second reporter nucleotide sequenceshybridize to form a double-stranded hybrid.

In certain embodiments, the first reporter nucleotide sequence iscovalently bonded to the target-binding component. The second reporternucleotide sequence and dye are subsequently added to form the mixture.The order in which the second reporter nucleotide sequence and dye areadded is not critical.

Alternatively, the first reporter nucleotide sequence and secondreporter nucleotide sequence can be covalently linked in a 5′-3′arrangement to form a self-hybridizing hairpin, or can be crosslinked.The hairpin or crosslinking stabilizes hybrid formation, and minimizesloss of single-stranded polynucleotide. In this embodiment, the dye isthen added to form the target binding complex. In another alternative,the dye can be covalently bonded to the first reporter nucleotidesequence or the second reporter nucleotide sequence.

In certain embodiments, the first reporter nucleotide sequence and/orthe second reporter nucleotide sequence can have a non-complementaryoverhang. It will be understood that the first and second reporternucleotide sequences are complementary while still maintaining one ormore overhangs.

The components of the reporter complex can be covalently linked to thetarget binding component by any means known in the art. The functionalgroups of amino acids of ligands suitable for covalent binding undermild conditions include but are not limited to (i) the alpha aminogroups of the chain and the epsilon amino groups of lysine and arginine,(ii) the alpha carboxyl group of the chain end and the beta and gammacarboxyl groups of aspartic and glutamic acids, (iii) the phenol ring oftyrosine, (iv) the thiol group of cysteine, (v) the hydroxyl groups ofserine and threonine, (vi) the imidazile group of histidine, and (vii)the indole group of tryptophan. Similarly, the nucleic acid analog canbe synthesized to contain any of these chemical moieties.

Those skilled in the art will recognize any number of establishedchemical conjugating techniques for covalently attaching nucleic acidanalogs to ligands. Some non-limiting examples include cyanogen bromideformation of reactive cyclic-imido carbamate for covalent coupling ofamines, carbodiimide formation of O-acyl isourea for coupling of amines,maleimidobenzoyl NHS ester formation for coupling of amines andsulfhydryls, and maleimidocaproic acid hydrazide HCl for coupling ofsulfhydryls and carbohydrates. Non-covalent attachments can beaccomplished by creating oxidized disulfide bonds between cysteines fromnucleic acid analogs and ligands. These can be easily reduced bydithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP) therebyreleasing the nucleic acid analog/polynucleotide hybrid from the ligandas needed.

The target binding component can be covalently attached to the at leastone component of the reporter complex by a linking group. Linking groupsare chemical moieties that link or connect reactive groups to ITPs. Thelinking group can be any linking group can include one or more alkylgroups such as methyl, ethyl, propyl, butyl, etc. groups, alkoxy groups,alkenyl groups, alkynyl groups or amino group substituted by alkylgroups, cycloalkyl groups, polycyclic groups, aryl groups, polyarylgroups, substituted aryl groups, heteroaryl groups, and substitutedheteroaryl groups. Linking groups may also comprise poly ethoxyaminoacids such as AEA ((2-amino) ethoxy acetic acid) or a preferredlinking group AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid).

The sequence of the first and second reporter nucleotide sequences maybe designed in a variety of ways. By way of example and not limitation,different reporter nucleotide sequence sequences may be optimized toproduce an increased rate of change in optical property of a sample thatincludes the dye. Empirically determined “universal” first and secondreporter nucleotide sequences will be used to maximally meet assayrequirements.

A series of first and second reporter nucleotide sequences can beprepared by any method known in the art. For example, the reporternucleotide sequences can be synthesized in vitro or in vivo (such as byrecombinant methods). After the reporter nucleotide sequences areannealed, the rate of change of the sample that includes the dye can betested under a number of different conditions. The greater the rate ofchange for first and second reporter nucleotide sequences have aspecific sequence, the when the first and second reporter nucleotidesequences are covalently linked in a 5′ to 3′ arrangement.

The first and second reporter nucleotide sequence sequences can be ofany length, provided that they hybridize together. The length of thefirst and second reporter nucleotide sequences also can be optimized, asdiscussed above.

The optimization methods can be adapted for any embodiment of firstreporter nucleotide sequence, second reporter nucleotide sequence, anddye. For example, the first or second reporter nucleotide sequences canbe covalently bonded to the target binding component, and the assay rateof change in the assay can be determined. The dye can be covalentlybound to the target binding component.

It will be understood that screening methods can be optimized for theaddition of any compound disclosed in the present invention.

B. Target binding components

The target binding component may be any molecule that is capable ofselectively interacting with a desired target. Exemplary targetsinclude, but are not limited to, cells, microorganisms (such asbacteria, fungi, and viruses), polypeptides, nucleic acids (such aspolynucleotides, cDNA molecules, or genomic DNA fragments), hormones,cytokines, drug molecules, carbohydrates, pesticides, dyes, amino acids,or small organic or inorganic molecules. Target binding componentshaving limited cross-reactivity are generally preferred. Exemplarytarget binding components include, for example, antibodies, antibodyfragments, non-antibody receptor molecules, template imprintedmaterials, lectins, enzymes, and organic or inorganic binding elements.

Some of the specific embodiments of target binding components areexplained in more detail below. This disclosure does not limit the scopeof the target binding components, as used herein.

1. Antibodies and Antibody Fragments

In certain embodiments, the target binding component may be an antibodyor an antibody fragment. For example, target binding components may bemonoclonal antibodies, or derivatives or analogs thereof, includingwithout limitation: Fv fragments, single chain Fv (scFv) fragments, Fab′fragments, F(ab′)₂ fragments, single domain antibodies, camelizedantibodies and fragments thereof, humanized antibodies and fragmentsthereof, and multivalent versions of the foregoing. Multivalent targetbinding components include without limitation: monospecific orbispecific antibodies, such as disulfide stabilized Fv fragments, scFvtandems ((ScFV)₂ fragments), diabodies, tribodies or tetrabodies, whichtypically are covalently linked or otherwise stabilized (e.g., leucinezipper or helix stabilized) scFv fragments; receptor molecules thatnaturally interact with a desired target molecule.

In one embodiment, the target binding component is preferably anantibody. Preparation of antibodies may be accomplished by any number ofwell-known methods. For generating monoclonal antibodies, presuming thatthe antigen of interest is known and available, the first step isimmunization of animals, typically mice, with a desired antigen (e.g., adesired target molecule or fragment thereof). Once the mice have beenimmunized, and preferably boosted one or more times with the desiredantigen(s), monoclonal antibody-producing hybridomas are preferablyprepared and screened according to well-known methods (see, e.g., Kuby,Janis, IMMUNOLOGY, Third Edition, pp. 131-139, W.H. Freeman & Co.(1997), for a general overview of monoclonal antibody production, thatportion of which is incorporated herein by reference).

In vitro methods that combine antibody recognition and phage displaytechniques allow one to amplify and select antibodies with very specificbinding capabilities. See, e.g., Holt et al., Current Opinion inBiotechnology 11:445 (2000). These methods typically are much lesscumbersome than preparation of hybridomas by traditional monoclonalantibody preparation methods. Binding epitopes may range in size fromsmall organic compounds such as bromo uridine and phosphotyrosine tooligopeptides on the order of 7-9 amino acids in length.

In another embodiment, the target binding component may be an antibodyfragment. Preparation of antibody fragments may be accomplished by anynumber of well-known methods. In one embodiment, phage displaytechnology may be used to generate antibody fragment target bindingcomponents that are specific for a desired target molecule, including,for example, Fab fragments, Fv's with an engineered intermoleculardisulfide bond to stabilize the VH-VL pair, scFvs, or diabody fragments.As an example, production of scFv antibody fragments using phage displayis described below.

For phage display, an immune response to a selected immunogen iselicited in an animal (such as a mouse, rabbit, goat or other animal)and the response is boosted to expand the immunogen-specific B-cellpopulation. Messenger RNA is isolated from those B-cells, or optionallya monoclonal or polyclonal hybridoma population. The mRNA isreverse-transcribed by known methods using either a poly-A primer ormurine immunoglobulin-specific primer(s), typically specific tosequences adjacent to the desired VH and VL chains, to yield cDNA. Thedesired VH and VL chains are amplified by polymerase chain reaction(PCR) typically using VH and VL specific primer sets, and are ligatedtogether, separated by a linker. VH and VL specific primer sets arecommercially available, for instance from Stratagene, Inc. of La Jolla,Calif.

Assembled VH-linker-VL product (encoding an scFv fragment) is selectedfor and amplified by PCR. Restriction sites are introduced into the endsof the VH-linker-VL product by PCR with primers including restrictionsites and the scFv fragment is inserted into a suitable expressionvector (typically a plasmid) for phage display. Other fragments, such asa Fab′ fragment, may be cloned into phage display vectors for surfaceexpression on phage particles. The phage may be any phage, such aslambda, but typically is a filamentous phage, such as fd and M1 3,typically M13.

In phage display vectors, the VH-linker-VL sequence is cloned into aphage surface protein (for M13, the surface proteins g3p (pIII) or g8p,most typically g3p). Phage display systems also include phagemidsystems, which are based on a phagemid plasmid vector containing thephage surface protein genes (for example, g3p and g8p of M1 3) and thephage origin of replication. To produce phage particles, cellscontaining the phagemid are rescued with helper phage providing theremaining proteins needed for the generation of phage. Only the phagemidvector is packaged in the resulting phage particles because replicationof the phagemid is grossly favored over replication of the helper phageDNA. Phagemid packaging systems for production of antibodies arecommercially available. One example of a commercially available phagemidpackaging system that also permits production of soluble ScFv fragmentsin bacteria cells is the Recombinant Phage Antibody System (RPAS),commercially available from Amersham Pharmacia Biotech, Inc. ofPiscataway, N.J. and the pSKAN Phagemid Display System, commerciallyavailable from MoBiTec, LLC of Marco Island, Fla. Phage display systems,their construction and screening methods are described in detail in,among others, U.S. Pat. Nos. 5,702,892, 5,750,373, 5,821,047, and6,127,132.

Typically, once phage are produced that display a desired antibodyfragment, epitope specific phage are selected by their affinity for thedesired immunogen and, optionally, their lack of affinity to compoundscontaining certain other structural features. A variety of methods maybe used for physically separating immunogen-binding phage fromnon-binding phage. Typically the immunogen is fixed to a surface and thephage are contacted with the surface. Non-binding phage are washed awaywhile binding phage remain bound. Bound phage are later eluted and areused to re-infect cells to amplify the selected species. A number ofrounds of affinity selection typically are used, often increasinglyhigher stringency washes, to amplify immunogen binding phage ofincreasing affinity. Negative selection techniques also may be used toselect for lack of binding to a desired target. In that case, un-bound(washed) phage are amplified.

Although it is preferred to use spleen cells and/or B-lymphocytes fromanimals pre-immunized with a desired immunogen as a source of cDNA fromwhich the sequences of the VH and VL chains are amplified by RT-PCR,naive (un-immunized with the target immunogen) splenocytes and/orB-cells may be used as a source of cDNA to produce a polyclonal set ofVH and VL chains that are selected in vitro by affinity, typically bythe above-described phage display (phagemid) method. When naive B-cellsare used, during affinity selection, the washing of the first selectionstep typically is of very low stringency so as to avoid loss of anysingle clone that may be present in very low copy number in thepolyclonal phage library. By this naive method, B-cells may be obtainedfrom any polyclonal source. B-cell or splenocyte cDNA libraries also area source of cDNA from which the VH and VL chains may be amplified. Forexample, suitable murine and human B-cell, lymphocyte and splenocytecDNA libraries are commercially available from Stratagene, Inc. and fromClontech Laboratories, Inc. of Palo Alto, Calif. Phagemid antibodylibraries and related screening services are provided commercially byCambridge Antibody Technology of the U.K. or MorphoSys USA, Inc.) ofCharlotte, N.C.

The target binding components do not have to originate from biologicalsources, such as from naive or immunized immune cells of animals orhumans. The target binding components may be screened from acombinatorial library of synthetic peptides. One such method isdescribed in U.S. Pat. No. 5,948,635, which described the production ofphagemid libraries having random amino acid insertions in the pIII geneof M1 3. These phage may be clonally amplified by affinity selection asdescribed above.

Panning in a culture dish or flask is one way to physically separatebinding phage from non-binding phage. Panning may be carried out in 96well plates in which desired immunogen structures have been immobilized.Functionalized 96 well plates, typically used as ELISA plates, may bepurchased from Pierce Biotechnology, Inc. of Rockford, Ill. Polypeptideimmunogens may be synthesized directly on NH₂ or COOH functionalizedplates in an N-terminal to C-terminal direction. Other affinity methodsfor isolating phage having a desired specificity include affixing theimmunogen to beads. The beads may be placed in a column and phage may bebound to the column, washed and eluted according to standard procedures.Alternatively, the beads may be magnetic so as to permit magneticseparation of the binding particles from the non-binding particles. Theimmunogen also may be affixed to a porous membrane or matrix, permittingeasy washing and elution of the binding phage.

In certain embodiments, it may be desirable to increase the specificityof the target binding component for a given target molecule using anegative selection step in the affinity selection process. For example,target binding component displaying phage may be contacted with asurface funtionalized with immunogens distinct from the target molecule.Phage are washed from the surface and non-binding phage are grown toclonally expand the population of non-binding phage thereby de-selectingphage that are not specific for the desired target molecule. Incertain-embodiments, random synthetic peptides may be used in thenegative selection step. In other embodiments, one or more immunogenshaving structural similarity to the target molecule may be used in thenegative selection step. For example, for a target molecule comprising apolypeptide, structurally similar immunogens may be polypeptides havingconservative amino acid substitutions, including but not limited to theconservative substitution groups such as: (i) a charged group,consisting of Glu, Asp, Lys, Arg, and H is, (ii) a positively-chargedgroup, consisting of Lys, Arg, and H is, (iii) a negatively-chargedgroup, consisting of Glu and Asp, (iv) an aromatic group, consisting ofPhe, Tyr, and Trp, (v) a nitrogen ring group, consisting of His and Trp,(vi) a large aliphatic nonpolar group, consisting of Val, Leu, and Ile,(vii) a slightly polar group, consisting of Met and Cys, (viii) asmall-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu,Gln, and Pro, (ix) an aliphatic group consisting of Val, Leu, Ile, Met,and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.Conservative substitutions also may be determined by one or moremethods, such as those used by the BLAST (Basic Local Alignment SearchTool) algorithm, such as a BLOSUM Substitution Scoring Matrix, such asthe BLOSUM 62 matrix, and the like. A functional way to define commonproperties between individual amino acids is to analyze the normalizedfrequencies of amino acid changes between corresponding proteins ofhomologous organisms (Schulz and Schirmer, PRINCIPLES OF PROTEINSTRUCTURE: Springer Advanced Texts in Chemistry, Springer-Verlag, NewYork, 1990).

Screening of target binding components will best be accomplished by highthroughput parallel selection, as described in Holt et al.Alternatively, high throughput parallel selection may be conducted bycommercial entities, such as by Cambridge Antibody Technologies orMorphoSys USA, Inc.

Alternatively, selection of a desired target bindingcomponent-displaying phage may be carried out using the followingmethod.

Step 1: Affinity purify phage under low stringency conditions for theirability to bind to an immunogen fixed to a solid support (for instance,beads in a column).

Step 2: Elute the bound phage and grow the eluted phage. Steps 1 and 2may be repeated with more stringent washes in Step 1.

Step 3: Absorb the phage under moderate stringency with a given proteinmixture digested with a proteolytic agent of interest. Wash away theunbound phage with a moderately stringent wash and grow the washedphage. Step 3 may be repeated with less stringent washes.

Step 4: Affinity purify phage under high stringency for their ability tobind to the immunogen fixed to a solid support. Elute the bound phageand grow the eluted phage.

Step 5: Plate the phage to select single plaques. Independently growphage selected from each plaque and confirm the specificity to thedesired immunogen.

This is a general guideline for the clonal expansion ofimmunogen-specific target binding components. Additional steps ofvarying stringency may be added at any stage to optimize the selectionprocess, or steps may be omitted or re-ordered. One or more steps may beadded where the phage population is selected for its inability to bindto other immunogens by absorption of the phage population with thoseother immunogens and amplification of the unbound phage population. Thatstep may be performed at any stage, but typically would be performedafter step 4.

In certain embodiments, it may be desirable to mutate the binding regionof the target binding component and select for target binding componentswith superior binding characteristics as compared to the un-mutatedtarget binding component. This may be accomplished by any standardmutagenesis technique, such as by PCR with Taq polymerase underconditions that cause errors. In such a case, the PCR primers could beused to amplify scFv-encoding sequences of phagemid plasmids underconditions that would cause mutations. The PCR product may then becloned into a phagemid vector and screened for the desired specificity,as described above.

In other embodiments, the target binding components may be modified tomake them more resistant to cleavage by proteases. For example, thestability of the target binding components of the present invention thatcomprise polypeptides may be increased by substituting one or more ofthe naturally occurring amino acids in the (L) configuration withD-amino acids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%,80%, 90% or 100% of the amino acid residues of the target bindingcomponents may be of the D configuration. The switch from L to D aminoacids neutralizes the digestion capabilities of many of the ubiquitouspeptidases found in the digestive tract. Alternatively, enhancedstability of the target binding components of the invention may beachieved by the introduction of modifications of the traditional peptidelinkages. For example, the introduction of a cyclic ring within thepolypeptide backbone may confer enhanced stability in order tocircumvent the effect of many proteolytic enzymes known to digestpolypeptides in the stomach or other digestive organs and in serum. Instill other embodiments, enhanced stability of the target bindingcomponents may be achieved by intercalating one or more dextrorotatoryamino acids (such as, dextrorotatory phenylalanine or dextrorotatorytryptophan) between the amino acids of the target binding component. Inexemplary embodiments, such modifications increase the proteaseresistance of the target binding components without affecting theiractivity or specificity of interaction with a desired target molecule.

In certain embodiments, the antibodies or variants thereof, may bemodified to make them less immunogenic when administered to a subject.For example, if the subject is human, the antibody may be “humanized”;where the complementarity determining region(s) of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al., NATURE 321:522 (1986), Tempest etal. BIOTECHNOLOGY 9:266 (1991), and U.S. Pat. No. 6,407,213. Also,transgenic mice, or other mammals, may be used to express humanizedantibodies. Such humanization may be partial or complete.

In another embodiment, the target binding component is a Fab fragment.Fab antibody fragments may be obtained by proteolysis of animmunoglobulin molecule using the protease papain. Papain digestionyields two identical antigen-binding fragments, termed “Fab fragments”,each with a single antigen-binding site, and a residual “Fc fragment”.In an exemplary embodiment, papain is first activated by reducing thesulfhydryl group in the active site with cysteine, mercaptoethanol ordithiothreitol. Heavy metals in the stock enzyme may be removed bychelation with EDTA (2 mM) to ensure maximum enzyme activity. Enzyme andsubstrate are normally mixed together in the ratio of 1:100 by weight.After incubation, the reaction can be stopped by irreversible alkylationof the thiol group with iodoacetamide or simply by dialysis. Thecompleteness of the digestion should be monitored by SDS-PAGE and thevarious fractions separated by protein A-Sepharose or ion exchangechromatography.

In still another embodiment, the target binding component is an F(ab′)₂fragment. F(ab′)₂ antibody fragments may be prepared from IgG moleculesusing limited proteolysis with the enzyme pepsin. Exemplary conditionsfor pepsin proteolysis are 100 times antibody excess w/w in acetatebuffer at pH 4.5 and 37° C. Pepsin treatment of intact immunoglobulinmolecules yields a F(ab′)₂ fragment that has two antigen-combining sitesand is still capable of crosslinking antigen. Fab′ antibody fragmentsmay be obtained by reducing F(ab′)₂ fragments using mercaptoethylamine.The Fab′ fragments may be separated from unsplit F(ab′)₂ fragments andconcentrated by application to a Sephadex G-25 column (M=46,000-58,000).

2. Non-Antibody Embodiments

In other embodiments, the target binding component may be a non-antibodyreceptor molecule, including, for example, receptors that naturallyrecognize a desired target molecule, receptors that have been modifiedto increase their specificity of interaction with a target molecule,receptor molecules that have been modified to interact with a desiredtarget molecule not naturally recognized by the receptor, and fragmentsof such receptor molecules (see, e.g., Skerra, MOLECULAR RECOGNITION13:167 (2000)).

3. Template Imprinting Materials

In other embodiments, the target binding components may be a templateimprinted material. Template imprinted materials are structures thathave an outer sugar layer and an underlying plasma-deposited layer. Theouter sugar layer contains indentations or imprints that arecomplementary in shape to a desired target molecule or template so as toallow specific interaction between the template imprinted structure andthe target molecule to which it is complementary. Template imprintingcan be utilized on the surface of a variety of structures, including,for example, medical prostheses (such as artificial heart valves,artificial limb joints, contact lenses and stents), microchips(preferably silicon-based microchips), and components of diagnosticequipment designed to detect specific microorganisms, such as viruses orbacteria.

Template-imprinted materials are discussed in U.S. Pat. No. 6,131,580.

In another embodiment, a target binding component of the invention maybe modified so that its rate of traversing the cellular membrane isincreased. For example, the target binding component may be attached toa peptide that promotes “transcytosis,” e.g., uptake of a polypeptide bycells. The peptide may be a portion of the HIV transactivator (TAT)protein, such as the fragment corresponding to residues 37-62 or 48-60of TAT, portions which have been observed to be rapidly taken up by acell in vitro (Green and Loewenstein, CELL 55:1179 (1989)).

Alternatively, the internalizing peptide may be derived from theDrosophila antennapedia protein, or homologs thereof. The 60 amino acidlong homoeodomain of the homeo-protein antennapedia has beendemonstrated to translocate through biological membranes and canfacilitate the translocation of heterologous polypeptides to which it iscoupled. Thus, target binding components may be fused to a peptideconsisting of about amino acids 42-58 of Drosophila antennapedia orshorter fragments for transcytosis (Derossi et al., J. BIOL. CHEM.271:18188 (1996); Derossi et al., J. BIOL. CHEM. 269:10444 (1994); andPerez et al., J. CELL Sci. 102:717 (1992). The transcytosis polypeptidemay also be a non-naturally-occurring membrane-translocating sequence(MTS), such as the peptide sequences disclosed in U.S. Pat. No.6,248,558.

In exemplary embodiments, the dissociation constant of the targetbinding component for a target molecule is optimized to allow real timemonitoring of the presence and/or concentration of the analyte in agiven patient, sample, or environment.

4. Lectins

The target binding component can be a lectin. Lectins are a class ofcarbohydrate-binding proteins found in plants, viruses, microorganismsand animals. Frequently, lectins are multimeric having two or more ofnon-covalently associated subunits. A lectin may contain two or more ofthe same subunit, such as Con A, or different subunits, such asPhaseolus vulgaris agglutinin. At least one component of the reportercomplex can be covalently bonded to the lectin.

Because of the specificity that each lectin has toward a particularcarbohydrate structure, oligosaccharides with identical sugarcompositions can be distinguished or separated. Certain lectins willbind only to structures with mannose or glucose residues, while othersmay recognize only galactose residues. Certain other lectins requirethat the particular sugar be in a terminal non-reducing position in theoligosaccharide, while others can bind to sugars within theoligosaccharide chain. Some lectins do not discriminate between a and banomers, while others require not only the correct anomeric structure,but a specific sequence of sugars for binding. The affinity between alectin and its receptor may vary a great deal due to small changes inthe carbohydrate structure of the receptor. All of these properties thatare peculiar to lectins enable one to discriminate between structures,to isolate one glycoconjugate, cell, or virus from a mixture, or tostudy one process among several. Because virtually all biologicalmembranes and cell walls contain glycoconjugates, all living organismscan be studied with lectins.

In certain embodiments, a target binding component can include achemical handle that facilitates its isolation, immobilization,identification, or detection, additionally, or in the alternative, thechemical handle can serve to increase the solubility of the targetbinding component. In various embodiments, chemical handles may be apolypeptide, a polynucleotide, a carbohydrate, a polymer, or a chemicalmoiety, or combinations or variants thereof. In certain embodiments,exemplary chemical handles include glutathione S-transferase (GST),protein A, protein G, calmodulin-binding peptide, thioredoxin, maltosebinding protein, HA, myc, poly arginine, poly H is, poly His-Asp or FLAGtags. Additional exemplary chemical handles include polypeptides thatalter protein localization in vivo, such as signal peptides, type IIIsecretion system-targeting peptides, transcytosis domains, nuclearlocalization signals, and the like. In various embodiments, a targetbinding component of the invention may include one or more chemicalhandles, including multiple copies of the same chemical handle or two ormore different chemical handles. It is also within the scope of theinvention to include a linker (such as a polypeptide sequence or achemical moiety) between a target binding component of the invention andthe chemical handle in order to facilitate construction of the moleculeor to optimize its structural constraints. In another embodiment, thetarget binding complex including a chemical handle may be constructed soas to contain protease cleavage sites between the chemical handle andthe target binding component of the invention in order to remove thechemical handle. Examples of suitable endoproteases for removal of achemical handle include Factor Xa and TEV proteases.

In other embodiments, the target binding component can be a drug, aputative drug, a drug target, or a putative drug target. Drugs aredisclosed, for example, in the MERCK INDEX 13^(th) Ed. (2001).

It will be understood that the target binding components discussed aboveare only exemplary. Any other target binding component that binds atarget can be used.

VIII. COMPOSITIONS

The present invention also contemplates compositions includingcomponents used in the methods disclosed herein.

In certain embodiments, the compositions include at least two of thecomponents that can be used in the methods disclosed herein. By way ofexample and not limitation, the composition can include a dye and aspecific nucleic acid analog. More specially, the composition caninclude a dye, and at least one of a chiral PNA, an LNA, a morpholinonucleic acid, a TNA, or a metal-linked nucleic acid. Such compositionscan further include a surfactant and/or a target. In certaincircumstances, alcohol is added in combination with a surfactant.

In other embodiments, the composition can include a detergent and atleast one of the other components used in the method. For example, acomposition can include a detergent and a nucleic acid analog, a dye,and/or a target polynucleotide. The detergent can be any detergent knownin the art. In certain formulations, the detergent can be a cationic,anionic, non-ionic, or zwitterionic detergent. In certain formulations,the detergent can be at least one of TMAC, LSS, SDS, Tween® 20, Tween®40, Tween® 80, NP40 Tergitol®, Span® 20, Span® 80, and CHAPS. In certainother formulations, the detergent can be at least one of Tween® 20,Tween® 40, Tween® 80, Tergitol® NP40, LSS, TMAC, and CHAPS. Thecomposition can also include a PNA, an LNA, a morpholino nucleic acid, aTNA, or a metal-linked nucleic acid. The composition can includemethanol, ethanol, isopropanol, butanol or other organic solvents knownin the art.

The composition can also include a dye combined with a targetpolynucleotide and nucleic acid analog. For example, the composition caninclude a target polynucleotide/nucleic acid analog hybrid, combinedwith a dye. The composition can further include one or more of any othercomponent disclosed in the methods herein.

The composition can also include a reagent that stops further change inthe optical property of the dye. By way of example and not limitation,the stopping reagent can be a solvent such as 20% methanol, 15%isopropanol, 15% DMSO, or 10% butanol. If a surfactant is present forexample, Tween® 80 at a concentration of about 0.05%, then a higherconcentration of solvents is needed, such as in the range of about40-50% methanol.

The composition can also include reagents such as 5 mM phosphateadjusted to pH 5.5. The composition can also include buffers such as 10mM Homopipes, pH 5.0 with 0.05% Tween 80, or 1×TE with 0.05% Tween 80.

In certain embodiments, a solid support with a specific background colorprovides significantly improved detection of the target polynucleotide.Compositions can include a solid surface with any color background,including a white background. The solid support, for example, can be amicrotiter plate having a white background. Alternatively, thecompositions can include a sample surface modified to have the sameinterior as any plate described herein.

The compositions can include any other compound, compounds, or deviceused in the methods disclosed herein, in any combination.

IX. KITS

In one aspect, the present invention provides a kit for detecting targetpolynucleotides. A kit may include one or more reagents useful in themethods or compositions disclosed herein. For example, kits can includedyes, nucleic acid analogs (immobilized or not), surfactants, sources oflight stimulus, buffers, alcohols, standards used for controls, keysillustrating positives and negatives of control samples for interpretingreaction results, and instructions. The kits may further includesuitable packaging of the respective compositions and/or other optionalcomponents as disclosed below. The specific components may be providedin suitable containers.

A. Dyes

The kits provided herein include one or more dyes. The dyes can includeany dye disclosed herein. The dyes can be provided in pre-packagesamounts, or can be provided in a single tube from which aliquots can beapportioned or diluted and then apportioned. The dyes may be furtherpackaged in any suitable packaging for segregation from other componentsof the kit and to facilitate dispensing of the composition.

B. Nucleic Acid Analogs

The kits may also include one or more nucleic acid analogs. The nucleicacid analog may be any nucleic acid analog, as described herein. Thenucleic acid analog may have any sequence that is complementary or fullycomplementary to a target nucleic acid sequence. The sequence may be anysequence known in the art. In one embodiment, the nucleic acid analoghas a sequence disclosed herein.

In one embodiment, the kit contains one or more nucleic acid analogprovided in any suitable container or containers (if the multiplenucleic acid analogs are packaged separately). The nucleic acidanalog(s) may be pre-aliquoted into usable amounts, or provided in asingle tube to be apportioned (with or without), or may be alreadyimmobilized on a solid surface. The container may be further packaged inany suitable packaging for segregation from other components of the kitand to facilitate dispensing aliquots. In another embodiment, two ormore the nucleic acid analog sequences may be contained in the samepackage. Nucleic acid analogs having differing sequences can be amixture in each tube, or they can be separately packaged two or moretubes, each with a single-sequence analog.

The kits may also include a vehicle to facilitate effectivehybridization of the nucleic acid analog to the target polynucleotide,such as a non-specific carrier polynucleotide, or other compound, suchas glycerol, or a vehicle that disrupts effective hybridization(possibly in the absence of surfactants), such as methanol, ethanol,butanol, DMSO, sodium hydroxide, and formamide.

C. Detergents

The kits preferably also include one or more detergents used in themethods and compositions disclosed herein. The detergent can be anydetergent known in the art. In certain other formulations, the detergentcan be a cationic, anionic, non-ionic, or zwitterionic surfactant. Incertain formulations, the surfactant can be at least one of TMAC, LSS,SDS, Tween® 20, Tween® 40, Tween® 80, NP40 Tergitol®, Span® 20, Span®80, and CHAPS. In still other formulations, the surfactant can be atleast one of Tween® 20, Tween® 40, Tween® 80, Tergitol® NP40, LSS, TMAC,Triton X100, Brij35, and CHAPS. The concentration of surfactant can beconcentrated such that mixtures that include the surfactant have aspecific concentration when diluted with other components.

The addition of alcohol to a mixture containing the detergent furtherreduces the photobleaching of the dye in the absence of a P/TP hybrid,but does not proportionally reduce the photobleaching of the dye in thepresence of a P/TP hybrid. The aspect of the present invention issurprising because, in the absence of detergent, the addition of alcoholcauses a greater photobleaching reduction in reactions with P/TP hybridthan in reactions without P/TP hybrid.

Any alcohol is preferably added so long as the added alcohol does notpreclude the hybridization of the NAA to its complement, or otherwisepreclude the catalytic activity of the NAA/NA hybrid. In certainembodiments, from about 8-12% ethanol or about 12-14% methanol ispreferably added in tandem with a detergent at about 0.05%-0.5%.

D. Source of Light Stimulus

The kits are preferably outfitted with a source of light stimulus. Thelight source is preferably any light source known in the art. The lightsource can be capable of adjusting intensity and/or wavelength.Non-limiting examples of light sources include the Sylvania Cool WhiteT8-CW, General Electric T8-C50, Fritz Aurora 50/50, a Sylvania dulux S9WCF₉DS/blue, Osram F9TT/50K, halogen autolamp, and SiC, InGaN, GaP,GaAsP, GaN+SiC, GaN-based Light Emitting Diodes (such as Jameco #183222a 470 nm LED, Jameco #334473 a 505 nm LED, Jameco #183214 a 515 nm LED,or a white multiwavelength (420-700 nm) LED #LLW5210200), or solid-statelasers.

E. Polynucleotide Manipulating Components

The kits may also include components used to manipulate or preservepolynucleotides, such as buffers, enzymes, columns, and other materials.

The buffers, enzymes, columns, and other materials can include thosethat are used to lyse cells or extract DNA or RNA from a cell. Thebuffers, enzymes, columns, and other materials can also includecomponents used to manipulate polynucleotides, including DNA and RNA.Such components include, for example, those disclosed in MOLECULARCLONING: A LABORATORY MANUAL, third edition (Sambrook et al, 2000) ColdSpring Harbor Press, or any other reference disclosed herein.

F. Instructions

Kits preferably include instructions for performing the methodsdescribed herein. Instructions may be included as a separate insertand/or as part of the packaging or container, e.g., as a label affixedto a container or as writing or other communication integrated as partof a container. The instructions may inform the user of methods forapplication and/or removal of the contents of the kit, precautions andmethods concerning handling of materials, expected results, warningsconcerning improper use, and the like.

G. Additional Optional Components of the Kits

Kits may further contain components useful in practicing the methodsdisclosed herein. Exemplary additional components includechemical-resistant disposal bags, tubes, diluent, gloves, scissors,marking pens and eye protection.

The compositions can also include any type of solid surface describedherein. The solid surface can be a specific color. In certaincircumstances, the solid surface is white. The solid surface may, forexample, be a microtiter plate having a number of white wells.

H. Computer Hardware and Software

The kits can also include computer hardware and/or computer softwarethat can be used to measure the optical property of the mixture or gelor surface where the dye is included or placed. The hardware can includeany detector used to measure the optical property. The software caninclude any algorithm used to note when a change in the optical propertyoccurs, or determine a rate of change in the optical property. The kitscan also include automated devices, such as those disclosed herein.

X. SOURCES OF TARGET POLYNUCLEOTIDES

The methods, compositions, and kits described herein have a variety ofuses. Non-limiting examples of these uses include detecting andquantifying organisms, including the subset thereof referred to aspathogens, toxins, and the like. Pathogens of interest that may bedetected using the present invention include foodborne pathogens,environmental pathogens, waterborne pathogens, or pathogens implicatedin bio- or agroterrorism. Other non-limiting uses include diseasediagnosis, such as sexually transmitted disease diagnosis, detection ofgenes conferring antibiotic resistance, detection of genes conferring apredisposition for drug responses, detection of genes implicated in aneffective drug response, detection of genetically-modified organisms,detection of non-indigenous flora or fauna, detection of specificcancer-related genes, and mRNA levels. Additional non-limitingapplications, relating, for example, to plant strain and/or grainquality, include agricultural applications and veterinary applications,many of which are the same or similar as the test developed for humans.

Examples, for illustration and not for limitation, are listed anddescribed in the U.S. Ser. No. 60/655,929 (“the '929 application”),which is incorporated herein in its entirety. In particular, the '929application sets forth detailed information regarding useful targetpolynucleotides, or descriptions of such polynucleotides, relating topathogens (pp. 50-57), host response polynucleotides (pp. 57-58),foodborne and environmental pathogens (pp. 58-61), waterborne pathogens(pp. 61-62), bio- and agroterrorism (pp. 62-63), disease diagnosticssuch as genetic diseases and cancers (pp. 64-69), sexually-transmitteddiseases (pp. 69-71), antibiotic resistance (pp. 71-72), geneticscreening for a predisposition for drug responses (pp. 72-73), genesimplicated in effective drug response (pp. 73-80), genetically-modifiedorganisms (p. 80), nonidigenous flora and fauna (pp. 80-81), andagricultural and/or veterinary applications (pp. 81-83).

One embodiment of the present invention relates to methods, materials,and kits directed at the detection the tuberculosis pathogens, includingMycobacterium tuberculosis. Nucleic acid analogs may be designed to havesequences or fragments of sequences similar or identical to PCR primersused to identify tuberculosis. Examples of these PCR primers aredisclosed in the art (see, e.g., M. J. Torres et al., DIAGN. MICROBIOL.INFECT. DIS. 45:207-12 (2003); B. Bhattacharya et al., TROP. MED. INT.HEALTH 8:150-7 (2003); M. Kafwabulula et al., INT. J. TUBERC. LUNG DIS.6:732-7 (2002)).

In another embodiment, nucleic acid analogs are designed that bind totarget polynucleotides common to an entire group of pathogens. Forexample, nucleic acid analogs may be designed to detect all bacteria(BP6) universal probe set, gram positive bacteria probe set (BP19), gramnegative bacteria probe set (BP3), and Fungi probe set (FP8). Anysequence in a set may be used. Examples of the sequences of the nucleicacid analogs for BP6, BP19, BP3, and FP8 are shown below. S=G and Cmixture, M=A and C mixture, Y=C and T mixture, and W=A and T mixtureaccording to IUB codes for mixed base sites.

TABLE 2 Pathogen SEQ ID Group Sequence NO: BP6 oI20185′ gaaSSMYcYaacacYtagcact 12 oI2019 5′ tacaaMgagYYgcWagacSgYgaS 13 BP19oI2021 5′ gcagYWaacgcattaagcact 14 oI2022 5′ acgacacgagctgacgacaa 15 BP3oI2003 5′ tctagctggtctgagaggatgac 16 oI2004 5′ gagttagccggtgcttcttct 17FP8 oI2055 5′ cctgcggcttaatttgactca 18 oI2057 5′ tagcgacgggcggtgtgta 19

The nucleic acid analogs can be used in clinical applications for thediagnosis of the microbial cause of sepsis or in other applicationswhere the microbial content of products in important in evaluating theirshelf-life and stability or other products where the sterility is beingassessed.

Target polynucleotides may be specific to ribosomal RNA sequences, suchas 16S RNA in E. coli. Ribosomal RNA contains specific sequences thatare characteristic to their organism. By using nucleic acid analogsequences that are complementary or exactly complementary to a targetpolynucleotide characteristic of the ribosomal RNA sequence, pathogensmay be identified based on their ribosomal RNA sequences. Ribosomal RNAsequences characteristic of different pathogens or strains of pathogens,may be found, for example, at D. J. Patel et al., J. MOL. BIOL.272:645-664 (1997).

The foregoing examples, and those incorporated by reference from the'181 application, are presented here to illustrate the breadth ofapplication for the present invention. Other examples of targetpolynucleotides usefully employed in the context of the presentinvention certainly exist, and more are identified daily as the naturalresult of scientific investigators attempting to understand the basisand develop cures for the many and various pathogens and diseases ofhumankind, plants, and animals. Additionally, target polynucleotidesthat measure the state of a locality's environment and other non-medicalor non-veterinary or non-agricultural applications are also contemplatedas usefully employed with the present invention.

EXAMPLES

The following non-limiting examples serve to more fully describe themanner of using the above-described methods and compositions. It isunderstood that these examples in no way serve to limit the scope of thesubject matter described herein, but rather are presented forillustrative purposes.

Throughout the examples, the term ‘smartDNA assay’ is used in referenceto an assay that employs the methods of the present invention.

All nucleic acid analog and DNA stock solutions in the followingexamples were made in 5 mM phosphate buffer, pH 5.5 unless otherwisenoted. The 5 mM phosphate buffer, pH 5.5, was used as the reactionbuffer in all examples, unless otherwise noted. The stock dye was madein methanol or DMSO.

Example 1

This example illustrates uses and effects of different detergents on thediagnostic method of the present invention.

The addition of different classes of detergents at differentconcentrations was shown to have different effects on the signal tonoise ratio. Some detergents increased the ratio while others had lessdramatic effects. In one embodiment, detergent added to the reactionbuffer increased the rate of change of the optical property in the test.In addition, each detergent reduced photobleaching of the dye innegative controls that lacked target polynucleotide.

Different detergents were added to the nucleic acid analog/targetpolynucleotide (P/TP) mixture, which included one of two targetpolynucleotides, one of two nucleic acid analogs, and a dye in a 5 mMphosphate buffer pH 5.5, at room temperature. Several different types ofdetergents were used, including cationic detergents, (specifically,tetramethyl ammonium chloride (“TMAC”)), anionic surfactants(specifically, N-lauroyl sarcosine sodium salt (“LSS”) and sodiumdodecyl sulfate (“SDS”)), nonionic detergents (specifically, variouspolyethylene glycol sorbitan monooleate solutions, sold under the tradenames Tween® 20, Tween® 40, and Tween® 80, a polyglycol ether detergentsold under the trade name Tergitol® NP-40; sorbitan monolaurate, soldunder the trade name Span® 20; and sorbitane monooleate, sold under thetrade name Span® 80), and zwitterionic detergents (specifically,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, commonlyknown by its acronym “CHAPS”). The various detergents were purchasedfrom Sigma-Aldrich, St. Louis, Mo. The term “detergent” is used hereinsynonymously with the term “surfactant”.

Aliquots of buffer reaction (each 5 mM phosphate, pH 5.5) containingdifferent surfactants were prepared, and P/TP mixtures with thesurfactants individually added were tested for the ability to (i)increase the sensitivity of the reaction and, separately, (ii) reducephotobleaching activity of the dye in a negative control. Eachexperiment contained four wells: (1) test well (non-chiral PNA, targetpolynucleotide, 3,3′-diethylthiacarbocyanine iodide dye and reactionbuffer, with surfactant); (2) negative control well (non-chiral PNA,non-targeted polynucleotide, 3,3′-diethylthiacarbocyanine iodide dye,and reaction buffer, with and without surfactant); (3) dye control well(3,3′-diethylthiacarbocyanine iodide dye and reaction buffer, with andwithout surfactant); and (4) buffer control well (50 μl of reactionbuffer, with and without surfactant). 10 pmoles 16S ncPNA (5′ACTGCTGCCTCCCGTAG 3′ [SEQ ID NO:8] or 5′ TGCCTCCCGTAG 3′ [SEQ ID NO:9]),10 pmoles of complementary oligo (5′CTACGGGAGGCAGCAGT 3′ [SEQ ID NO:10]or 5′CTACGGGAGGC 3′ [SEQ ID NO: 11]), and 4 nmoles of3,3′-diethylthiacarbocyanine iodide (“DTCC”; Sigma-Aldrich, St. Louis,Mo.) were placed in a 50 μl total reaction volume. ncPNAs (AppliedBiosystems, Foster City, Calif.) and oligos (Sigma Genosys, St. Louis,Mo.) were reconstituted in water that was both DNase- and RNase-free as100 μM stocks and further diluted to 2 μM working stocks. The DTCC dyewas dissolved in dimethyl sulfoxide (“DMSO”; Sigma-Aldrich, St. Louis,Mo.) as an 8 mM stock. This stock was further diluted to a 2 mM workingstock in 5 mM phosphate buffer (pH 5.5).

An initial fluorescence measurement was taken at time zero using a TecanGenios microplate reader with the wavelengths set at 535 nm (forexcitation) and 590 nm (for emission). The mixtures were then exposed toa light stimulus using the Aurora 50/50 (Fritz Industries, Inc.,Mesquite, Tex.) for 1 minute intervals with fluorescence readings takenafter each exposure for 5 minutes. For each reaction, the fluorescentemission values from the test well were compared to the fluorescentemission value from the negative control well (containing only ncPNA,dye, and reaction buffer) and converted to the percent changed. Thepercent change is calculated by the equation100−[(RFU_(TW))÷(RFU_(NC))]×100,where RFU_(TW) represents the fluorescent emission in relativefluorescent units of a reaction mixture in a test well that includes allrequired components of the test and RFU_(NC) represents the fluorescentemission in relative fluorescent units of a reaction mixture in anegative control well that does not include a target polynucleotide. Thepercent change is calculated at each time point. The percent differencebetween the measured rate in samples containing the targetpolynucleotide and the measured rate in samples not containing targetpolynucleotide was used to indicated the relative ability to detectpresence of the target polynucleotide sequence.

FIG. 1 graphically presents the data collected in the studies detailedhere. The reactions were each conducted in the presence of a detergenthaving a concentration of 0.05% v/v. Detergents added to the reactionbuffer included the nonionic detergents Tween® 20 (♦), Tween® 40 (⋄),Tween® 80 (◯), and Tergitol® NP-40 (●); the anionic detergent LSS (▬);the cationic detergent, TMAC (□); and the zwitterionic detergent CHAPS(x); where the symbols identified parenthetically after each of thenamed detergents are those used in the graphs of FIGS. 1 and 2. Improvedsensitivity resulted upon the addition of 0.05% of any of the detergentswhen compared to the absence of detergent added. Decreased change influorescent emission of the dye in the absence of a nucleic acidanalog/target polynucleotide hybrid compared to the dye alone wasobserved with the addition of Tween® 20, Tween® 40, Tween® 80, Tergitol®NP-40, LSS, TMAC and CHAPS. The rate of change was measured as thepercent change in fluorescence of the test well as compared to thenegative control in a concentration dependent manner.

Decreased change in fluorescent emission of the dye in the absence of anucleic acid analog/target polynucleotide hybrid compared to the dyealone was not observed when the concentration of detergent was increasedto 5.0%, however. FIG. 2 depicts the addition of detergents at aconcentration of 5.0% to the reaction buffer. Detergents added to thereaction buffer included the nonionic detergents Tween® 20 (♦), Tween®40 (⋄), Tween® 80 (◯), and Tergitol® NP-40 (●); the anionic detergentLSS (▪); the cationic detergent TMAC (□); and the zwitterionic detergentCHAPS (x). Increasing the detergent to 5.0% in the reaction buffersresulted in a smaller percent change in the fluorescent rate for allreactions except those that included detergents LSS and CHAPS.

Example 2

This example illustrates the usefulness of adding surfactants to thereaction buffer, which was sufficient for preparing samples for testing.

The addition of certain surfactants to the reaction buffer was found topermeabilize and/or lyse bacterial cells. Suprisingly, the assay fordetermining whether a particular target polynucleotide was present didnot require further purification of the so-permeabilized/lysed cells.

Reaction buffer with different surfactants was used aspermeabilization/hybridization buffer for bacteria to test whetherseparate nucleic acid isolation steps were necessary to perform thediagnostic method of the present invention. The addition of 0.5% Tween®20, Tween® 40, Tergitol® NP-40, N-lauryl sarcosine sodium salt (LSS), orCHAPS to the phosphate reaction buffer effectively permeabilized and/orlysed bacteria from cultures grown overnight in tryptic soy broth, asdemonstrated in the following experiment.

300 μl of overnight E. coli bacterial culture was centrifuged usingstandard procedures, resulting in a pellet of bacteria at the bottom ofthe tube. The supernatant was removed by aspiration. The pellet was thenre-suspended in 390 μl of reaction buffer (5 mM phosphate) or lysisbuffer (5 mM phosphate, pH 5.5, 0.05% surfactant) and incubated at roomtemperature for 10 minutes before an aliquot was used in the diagnosticreaction. 5 μl of the cells as resuspended in the two buffers wererespectively and separately combined with 10 pmoles of 16S ncPNA (5′-ACTGCT GCC TCC CGT AG-3′ [SEQ ID NO:8] or 5′-TGC CTC CCG TAG-3′ [SEQ IDNO:9]) and 4 nmoles of 3,3′-diethylthiacarbocyanine iodide (DTCC dye) ina 50 μl total reaction volume. In all cases tested, the reaction bufferwithout surfactant showed a reaction indicating the presence of a targetpolynucleotide, but it was slower and of a lesser extent of reactionthan were those samples subjected to the lysis buffer. Accordingly,further testing focused on use of the lysis buffer.

The data generated in these studies were used to create the graph ofFIG. 3. All reactions were conducted in reaction buffer with one of thesurfactants at a concentration of 0.5%, which surfactants were: Tween®20 (Δ), 0.5% Tween® 40 (⋄), 0.5% Tergitol® NP-40 (□), 0.05% laurylsarcosine salt (◯) or 0.05% CHAPS (*) in phosphate buffer. The positivecontrol (♦) used purified bacterial DNA in standard phosphate buffer;and the negative control (x) included all elements of the standardreaction apart from a target polynucleotide. A phosphate buffer control(▪) was included as well.

For each surfactant, the test well contained non-chiral PNA, targetnucleic acid, 3,3′-diethylthiacarbocyanine iodide dye and lysis buffer(with surfactant). The test well was compared to the negative controlthat contained non-chiral PNA, 3,3′-diethylthiacarbocyanine iodide dyeand lysis buffer (with surfactant). The phosphate buffer only test (E)contained non-chiral PNA, target polynucleotide,3,3′-diethylthiacarbocyanine iodide dye and reaction buffer (withoutsurfactant). The phosphate buffer only well was compared to the negativecontrol phosphate buffer that contained non-chiral PNA,3,3′-diethylthiacarbocyanine iodide dye and reaction buffer (withoutsurfactant).

An initial fluorescence reading was taken at time zero in the TecanGenios microplate reader with the wavelengths set at 535 nm excitationand 590 nm emission. The mixtures were then exposed to a light stimulususing the Aurora 50/50 for 1 minute intervals with fluorescence readingbeing taken after each exposure for 10 minutes. For each reaction, thefluorescent emission values from the test well were compared to thenegative control well and converted to the percent changed. The percentdifference between the measured rate in samples containing the targetnucleic acid and the rate in which the amount of target nucleic acid waszero indicated the presence of the target nucleic acid sequence. Thedifference corresponds to a relative decrease in fluorescence intensityof the test sample.

The presence of a target polynucleotide was detected in the presence ofcell lysate, without requiring additional purification.

FIG. 3 shows the percent change in fluorescence compared to thelysis/hybridization buffer containing only phosphate buffer. Thepresence of the target nucleic acid sequence was determined forsurfactants at 0.05% and 0.5% concentration. When light stimulus wasapplied, the rate of change in the fluorescence compared to the controlcorresponded to the presence of the target polynucleotide. The percentchange in fluorescence was a decrease in the fluorescence intensity ofthe dye.

Example 3

This example illustrates the effect of using different nucleic acidanalogs in the diagnostic test of the present invention.

Different nucleic acid analogs were used to determine the presence orquantity of nucleic acid in a sample. Chiral PNA molecules, LNAmolecules, and morpholino nucleic acid analogs were compared withnon-chiral PNA molecules.

The non-chiral PNA and the chiral PNA had the sequence 5′ TGC CTC CCGTAG 3′ [SEQ ID NO:9], where the phosphodiester bonded sugar backbone ofthe native polynucleotide were replaced with a peptide bondedpolypeptide backbone, as described further herein and well known withinthe art. Three LNA molecules designated LNA1, LNA2, and LNA3 were used,each having the same sequence of bases described here as SEQ ID NO:9,where one or more of the included nucleotides included a methylenebridge on their respective ribofuranose rings (forming a “locked”residue), as indicated: (1) LNA1 includes only locked residues; (2) LNA2and (3) LNA3 include a subset of locked residues identified by the uppercase letters at certain places on the sequence, as follows: 5′ TgC cTcCcG tAg 3′ for LNA2 and 5′ tGc cTc cCg tAg 3′ for LNA3. Morpholinonucleic acid analogs used here also had the base sequence of SEQ IDNO:9, formed from the analog nucleotides.

10 pmoles of nucleic acid analog, 10 pmoles of target polynucleotidehaving the sequence 5′CTA CGG GAG GCA 3′ [SEQ ID NO:12], and 4 nmoles of3,3′-diethylthiacarbocyanine iodide dye were placed in a 50 μl totalreaction volume to form a mixture. A negative control containing nucleicacid analog, 3,3′-diethylthiacarbocyanine iodide dye, reaction buffer,and a known (zero) amount of target polynucleotide was also tested. Allthe nucleic acid analogs and target polynucleotides were reconstitutedin DNase- and RNase-free water to 100 μM stock and further diluted to 2μM working stocks. The dye was dissolved in DMSO to generate an 8 mMstock. The 8 mM dye stock was further diluted to generate a 2 mM workingstock in 5 mM phosphate buffer (pH 5.5). The reaction buffer was a 5 mMphosphate buffer (pH 5.5).

An initial fluorescence reading was taken at time zero in the TecanGenios microplate reader with the wavelengths set at 535 nm forexcitation and 590 nm for emission. The mixtures were then exposed to a2000 foot-candle light stimulus using the Aurora 50/50 for 1 minuteintervals and measuring the fluorescence at one minute intervals for 5minutes. For each reaction, the fluorescent emission values from thetest well (nucleic acid analog, target polynucleotide, dye, and reactionbuffer) was compared to the negative control well (nucleic acid analog,dye and reaction buffer). The change in fluorescence was converted tothe percent changed and normalized to the negative control containingonly dye. The percent difference between the measured rate in samplescontaining the target nucleic acid and the rate in which the amount oftarget nucleic acid was zero indicated the presence of the targetnucleic acid sequence.

FIG. 4 displays a graph that is based on the data collected in the studyof this example and manipulated as indicated above. As can be seen inthe graph, the percent change in fluorescence intensity of each nucleicacid analog tested follows approximately the same profile of reaching inexcess of 80% reduction in fluorescence emission by three minutesexposure to the light stimulus. Mixtures including non-chiral PNA (▪),chiral PNA (□), LNA 1, 2, and 3 (respectively represented by x, ▬, *),and morpholino (Δ) nucleic acid analogs showed a difference in the rateof change in an optical property of the dye compared to when no nucleicacid analog was present in the mixture (i.e., the negative control(the * indicating 0% change)). Of the various nucleic acid analogstested, the chiral PNA may require more activation exposure in view ofthe percent change differences between mixtures including the chiral PNAand all others: after one and two minutes of light stimulus exposure,the cPNA mixture evidenced 30% and 65% changes in fluorescence,respectively; in contrast, the other reaction mixtures containing any ofthe other nucleic acid analogs evidenced at least 38% and 75% changes,respectively. Nonetheless, the data presented here supports theusefulness of all nucleic acid analogs tested for inclusion in thediagnostic method of the present invention.

Example 4

This example illustrates different approaches for identifying dyes thatare usefully employed in the context of the diagnostic method of thepresent invention.

In particular, this example presents data directed at determining ifselected dyes exhibit a photo-induced change in fluorescence orabsorbance based on results observed from light stimulus activation ofthe combinations of the respective dyes and: (1) a nucleic acid analog,namely a non-chiral PNA probe [SEQ ID NO:1], or (2) a targetoligonucleotide (“oligo”) that is exactly complementary to the PNAprobe; or (3) the hybridized combination of the two. Each of theseresults were compared to a negative control mixture lacking ncPNA andoligo.

All dyes tested were from Sigma (St. Louis, Mo.) except the following:3,3′-Diethylthiacyanine ethylsulfate (Organica),3-Ethyl-9-methyl-3′-(3-sulfatobutyl)thiacarbocyanine betaine (Organica),3-Carboxymethyl-3′,9-diethyl-5,5′-dimethylthiacarbocyanine betaine(Organica), 3,3′-Diallylthiacarbocyanine Bromide (Pfaltz and Bauer),3,3′-Diethyl-2,2′-Oxathiacarbocyanine Iodide (Pfaltz and Bauer),[5-[2-(3-Ethyl-3H-benzothiazol-2-ylidene)-ethylidene]-4-oxo-2-thioxo-thiazolidin-3-yl]-aceticacid (FEW),1-Butyl-2-[3-(1-butyl-1H-benzo[cd]indol-2-ylidene)-propenyl]-benzo[cd]indoliumtetrafluoroborate (FEW),5,6-Dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-benzimidazol-2-ylidene)-propenyl]-1,3-diethyl-3H-benzimidazoliumiodide, and d)1,3,3-Trimethyl-2-(2-[2-phenylsulfanyl-3-[2-(1,3,3-trimethyl-1,3-dihydro-indol-2-ylidene)-ethylidene]-cyclohex-1-enyl]-vinyl)-3H-indoliumchloride (FEW).

Xenon Protocol: In one test protocol, a xenon light source was used tophotoactivate reaction mixtures over a one minute period. Solutionscontaining concentrations of dye at 6 μM were prepared by diluting astock solution of 5 mM dye (in methanol) in buffer (5 mM PO₄) plussurfactant (0.05% Tween® 80). The dyes used to prepare the 6 μM dyesolutions are set forth in the summary of dyes studied that appearsbelow in Table 3.

For the Xenon protocol, a 48 μL aliquot of each 6 μM dye solution wasadded to four wells of a 384-well microtiter plate (Costar, #3705). Foreach dye: a 1 μL aliquot of 5 μM oligo [SEQ ID NO:20] was added to thefirst and second well, a 1 μL aliquot of 5 μM ncPNA [SEQ ID NO: 1] wasadded to the first and third well, a 1 μL aliquot of ddH₂O (Nanopure)was added to the second and third well, and a 2 μL aliquot of ddH₂O wasadded to the fourth well.

Fluorescent spectra at T₀ were obtained for each well using a TecanSafire2 microplate reader. Parameters for the spectral scanning were:excitation range of 300 to 652 nm with a resolution (step size) of 11nm, emission range of 375 to 723 nm with a resolution of 6 nm. The platewas then exposed to a 450W Xenon arc lamp (Ushio, #UXL-451-O) for 1minute of photoactivation and fluorescent spectra were then read asbefore. Under the conditions used, dyes that showed a difference in anoptical property of test reaction mixtures (containing ncPNA/targetoligo) compared to the same optical property of dye-only reactionmixtures are indicated by a “yes” in Table 3. Distinctions between typesof differences are not distinguished in Table 3.

Aurora Protocol: In a second protocol, the reaction mixtures wereexposed to the Aurora 50/50 light activation source and observed from 0to 30 minutes. Solutions containing concentrations of dye at 25 μM wereprepared by diluting stock solutions of 5 mM dye (in methanol) inmolecular biology grade water (Hyclone, catalog #SH30538.03). The dyesused to prepare the 25 μM dye solutions are set forth in the summary ofdyes studied that appears in Table 3.

For the Aurora Protocol, two different reaction mixtures were preparedfor each dye. The first mixture consisted of 1 mL of 25 μM dye in water.The second mixture was identical to the first with the exception that 1μL of a 50 μM ncPNA [SEQ ID NO: 1]/target oligo [SEQ ID NO:20] mixturewas added to the dye mixture. Spectral scans of the reaction mixtureswere done using a 1 mL quartz cuvette (against water in a secondreference cuvette) in a Shimadzu 160UV Spectrophotometer. A spectrumfrom 200 nm to 800 nm for each reaction mixture was taken prior toexposure to a light stimulus (a 15 watt Aurora 50/50 fluorescent bulb,Fritz Industries). Parafilm was wrapped around the top of the cuvettesto prevent spillage of the solutions when the cuvettes were placedhorizontally (length-wise) across the fluorescent light bulb. Thesolutions in the cuvettes were exposed to light for 5 minutes, followedby a spectral scan. This exposure-spectral scan cycle was repeated outto at least 10 minutes total light exposure. For each dye tested, theabsorbance at lambda max (for time zero) of each spectrum was plotted asa function of time to determine the rate of absorbance decay for thencPNA/target oligo mixture relative to the rate of absorbance decay forthe dye only mixture. Under the conditions used, dyes that showed adifference in an optical property of test reaction mixtures (containingncPNA/target oligo) compared to the same optical property of dye onlyreaction mixtures are indicated by a “yes” in Table 3. Distinctionsbetween types of differences are not distinguished in Table 3.

LED Protocol. In embodiment C, solutions containing variousconcentrations of dye were prepared by diluting stock solutions of 5 mMdye (in methanol or DMSO) in buffer (10 mM TE) with surfactant (0.1%Tween® 80) to a final concentration which provided an absorbance atlambda-max of 0.5-1.0 absorbance units (in a 50 μL volume in a 384-wellwhite/clear microplate [NUNC, #242763]. The dyes used to prepare the dyesolutions are set forth in the summary of dyes studied that appears inTable 3.

In this embodiment, four different reaction mixtures were prepared foreach dye. The test reaction mixture was prepared by adding 30 μL dyesolution, 10 μL of 500 nM ncPNA (in water) [SEQ ID NO:1] and 10 μL of500 nM target oligo (in water) [SEQ ID NO:20] to a first well. Thetarget oligo control reaction mixture was prepared by adding 30 μL dyesolution, 10 μL molecular biology grade water (Hyclone) and 10 μL 500 nMtarget oligo [SEQ ID NO:20] to a second well. The ncPNA (probe) controlreaction mixture was prepared by adding 30 μL dye solution, 10 μL of 500nM ncPNA [SEQ ID NO:1] and 10 μL molecular biology grade water to athird well. The dye only control reaction mixture was prepared by adding30 μL dye solution and 20 μL molecular biology grade water to a fourthwell. Using a Safire2 microplate reader, absorbance measurements weretaken at the lambda max of each dye tested. The plate was then removedfrom the reader and exposed to light from various banks of LEDs atvarious peak wavelengths. After 10 minutes photoactivation, the platewas removed from the light source and absorbance measurements were takenas previously. The plate was then removed from the reader, exposed tolight from the photoactivator for an additional 50 minutes, and finalabsorbance measurements were taken. The absorbance at lambda max (fortime zero, for each dye tested) was plotted against time to determinethe rate of absorbance decay for the test reaction mixture, for thetarget oligo control reaction mixture, for the ncPNA control reactionmixture, and for the dye only control reaction mixture. Under theconditions used, dyes which showed a difference in an optical propertyof test reaction mixtures (containing ncPNA/target oligo) compared tothe same optical property of dye only reaction mixtures are indicated bya “yes” in Table 3. Distinctions between types of differences are notdistinguished in Table 3.

TABLE 3 Xenon Aurora LED # Dye Protocol Protocol Protocol 13,3′-Dimethyloxacarbocyanine iodide Yes No No 2 3,3′-Diethylthiacyanineiodide Yes No No 3 3,3′-Diethylthiacyanine ethylsulfate ND No No 43,3′-Diethylthiacarbocyanine iodide Yes Yes Yes 53,3′-Diethyl-9-methylthiacarbocyanine Yes Yes Yes iodide 63-Ethyl-9-methyl-3′-(3-sulfatobutyl) ND No No thiacarbocyanine betaine 73-Carboxymethyl-3′,9-diethyl-5,5′- ND No No dimethylthiacarbocyaninebetaine 8 3,3′-Diethylthiadicarbocyanine iodide Yes No Yes 93,3′-Diethylthiatricarbocyanine iodide Yes No No 103,3′-Diethylthiatricarbocyanine Yes No No perchlorate 113,3′-Diethyloxacarbocyanine iodide Yes No No 123,3′-Diethyloxadicarbocyanine iodide Yes Yes No 133,3′-Dipropylthiacarbocyanine iodide Yes Yes Yes 143,3′-Dipropylthiadicarbocyanine No No No iodide 153,3′-Dipropyloxacarbocyanine iodide No No No 163,3′-Dibutylthiacarbocyanine iodide Yes Yes Yes 173,3′-Dipentylthiacarbocyanine iodide Yes Yes Yes 183,3′-Dihexyloxacarbocyanine iodide No No No 193,3′-Diallylthiacarbocyanine Bromide Yes Yes Yes 203,3′-Diethyl-2,2′-Oxathiacarbocyanine Yes No No Iodide 211,1′-Diethyl-2,2′-cyanine iodide Yes No No 221-1′-Diethyl-2,2′-carbocyanine iodide Yes Yes Yes 231,1′-Diethyl-2,2′-carbocyanine ND Yes Yes bromide 241,1′-Diethyl-4,4′-carbocyanine iodide No No No 251,1′-Diethyl-3,3,3′,3′- No No No tetramethylindocarbocyanine iodide 261,1′-Dipropyl-3,3,3′,3′- No No No tetramethylindocarbocyanine iodine 27[5-[2-(3-Ethyl-3H-benzothiazol-2- ND No Noylidene)-ethylidene]-4-oxo-2-thioxo- thiazolidin-3-yl]-acetic acid 281-Butyl-2-[3-(1-butyl-1H- ND No No benzo[cd]indol-2-ylidene)-propenyl]-benzo[cd]indolium tetrafluoroborate 295,6-Dichloro-2-[3-(5,6-dichloro-1,3- ND No NDdiethyl-1,3-dihydro-benzimidazol-2- ylidene)-propenyl]-1,3-diethyl-3H-benzimidazolium iodide 30 1,3,3-Trimethyl-2-(2-[2- ND No Nophenylsulfanyl-3-[2-(1,3,3-trimethyl- 1,3-dihydro-indol-2-ylidene)-ethylidene]-cyclohex-1-enyl]-vinyl)- 3H-indolium chloride 314,5,4′,5′-Dibenzo-3,3′-diethyl-9- ND No No methyl-thiacarbocyaninebromide

Of 31 dyes tested using the three different protocols set forth above,only six were shown to not change color; another eight have notdemonstrated positive results, but were not tested with all threeprotocols as yet and therefore remain inconclusive. Over half of thetotal number of dyes tested displayed positive results. Accordingly,placing the eight that are as yet incompletely tested to the side, theresults are currently indicating that 17 out of 23 fully tested dyesshow positive results, i.e., a 74% rate of success.

Example 5

A method of target Nucleic Acid (NA) elution that was previouslycaptured to PNA microparticles (Streptavidin based).

All steps were performed at room temperature. The solid state lightsource consisted of 192 2000-mcd 470 nm light emitting diodes (LED)(Jameco Electronics P/N 183222) arranged in a 5×7 inch rectangular arrayplaced 2 inches above the microplate surface. With a 15V power supply,this configuration produced an irradiance of approximately 2 mWcm⁻² atthe surface of a standard microplate. The effect of the voltage of thepower supply used with the light source on the elution was determinedand compared to elution on a benchtop (with ambient light) and in thedark. The light source was used with different voltage of powersuppliers, including 12V, 15V and 18V.

In this method, 20 pmole capture capacity of PNA microparticles wereused in reaction mixtures containing either 8 ng of MTB DNA and 16 ng ofhuman DNA (as the non-specific DNA), or containing 16 ng of human DNAonly, via a smartDNA capture assay.

The capture and elution procedures are all done in a 1.5 ml eppendorftubes.

In this assay the target NA was incubated with the PNA microparticlesfor 30 minutes with shaking, in 100 μl capture buffer: 10 mM Homopipesbuffer, pH 5, 18 μM DiSC₂(3), 0.1% Tween-80 and 0.5 mM EDTA. Afterwashing with wash buffer (10 mM Homopipes buffer, pH 5, 9 μM DiSC₂(3),0.05% Tween-80 and 0.5 mM EDTA) for 3 times (200 μl each time), theelution buffer (100 μl 1 mM EDTA) was added to contents of the tubes inlow light and subjected to one of five treatments:

1). Incubation in low light (dark room) for 30 minutes

2). Incubation on benchtop for 30 minutes

3). Exposed to LED 470 nm, 12V for 5 minutes

4). Exposed to LED 470 nm, 15V for 5 minutes

5). Exposed to LED 470 nm, 18V for 5 minutes

After the elution, the liquid (˜100 μl) (with no microparticles) wasaliquoted into triplicate wells in a microplate (each reaction contain25 μl of the eluate), and to each well, PNA and dye mixture (25 μl, with320 nM of PNA, 18 μM of DiSC₂(3), in 20 mM Homopipes buffer, pH 5, 0.1%Tween 80) was added. The mixture was then incubated for 10 minutes inthe dark room.

The initial dye color change rates were measured.

Results are summarized in Table 4 below.

TABLE 4 Initial dye color change rate (mAbsorbance units/minute) ofsamples after 5 different elution parameters DNA dark benchtop 12 V 15 V18 V MTB and 81 112 98 123 160 human human 18.3 33.5 22.2 31.4 42.9

Comparing with the standard elution procedure (benchtop for 30 min), theelution in the 15V LED box for 5 min resulted in a similar sensitivityand specificity. Thus 15V LED elution can be used as alternative tobenchtop elution, and can shorten the total time required to run theentire assay.

Example 6

This example illustrates one embodiment of a gel-based assay using thediagnostic test of the present invention.

The methods presented herein can be used to identify the presence of atarget polynucleotide in a gel-based assay. Complementary pairs of ncPNAand single-strand polynucleotides were mixed together for at least 30minutes at room temperature and loaded onto a 3% agarose gel. Theagarose gel contained 2.5 μM of DTCC. As shown in FIG. 7, the first fourlanes contained double- and single-stranded DNA size standards of 40base pairs (or 40 bases) each, as follows:

Lane 1 100 pmoles double-stranded DNA(5′-CCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAAT-3′ [SEQ ID NO: 48], pluscomplementary strand 5′-ATTGAGCGGGTTGATCCAAGAAAGGACCCGGTCGTCCTGG-3′ [SEQID NO: 49]);

Lane 2 200 pmoles double-stranded DNA (same as above);

Lane 3 100 pmoles single-stranded DNA(5′-TGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGT-3′ [SEQ ID NO: 50]; and

Lane 4 200 pmoles single-stranded DNA (same as above)

The remaining lanes contained different combinations of ncPNA and itsrespective complementary single-stranded target polynucleotidesequences, as follows: Lane 5, 100 pmoles ncPNA 19-mer having thefollowing sequence GTTGATCCAAGAAAGGACC-lysine [SEQ ID NO: 51] plus 100pmoles of single-stranded DNA 40-mer target polynucleotide having thefollowing sequence 5′-CCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAAT-3′ [SEQID NO: 48]; Lane 6, same as lane 5 except 200 pmoles of ncPNA 19-merplus 200 pmoles of single-stranded DNA 40-mer target polynucleotide;Lane 7, 100 pmoles ncPNA 21-mer having the following sequenceGTTGATCCAAGAAAGGACCCG-lysine [SEQ ID NO: 51] plus 100 pmolessingle-stranded DNA 40-mer target polynucleotide having the followingsequence 5′-CCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAAT-3′ [SEQ ID NO: 48];Lane 8, same as lane 7 except 200 pmoles of ncPNA and 200 pmoles ofsingle-stranded 40-mer DNA target; Lane 9, 100 pmoles ncPNA 17-merhaving the following sequence TTTCGCGACCCAACACT-lysine [SEQ ID NO: 53]plus 100 pmoles of single-stranded DNA 40-mer target polynucleotidehaving the following sequence5′-TGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGT-3′ [SEQ ID NO: 50]; Lane 10,same as lane 9 except 200 pmoles ncPNA 17-mer and 200 pmolessingle-stranded DNA 40-mer target polynucleotide; lane 11, 100 pmoles ofanother ncPNA 17-mer having the following sequenceAGTGTTGGGTCGCGAAA-lysine [SEQ ID NO: 55] plus 100 pmoles ofsingle-stranded DNA 40-mer target polynucleotide having the followingsequence 5′-ACCACAAGGCCTTTCGCGACCCAACACTACTCGGCTAGCA-3′ [SEQ ID NO: 56].

Electrophoresis was conducted in the 3% agarose/2.5 μM DTCC/1×TBE gel at200V for 20 minutes. After illuminating the gel with white light fromthe Aurora 50/50 for 5 minutes, the gel was observed. A 254 nm UVtransilluminator and B&W Polaroid camera were used to photograph the gelbefore and after photoactivation (upper and lower panels, respectively).The gel generally had a pink color with a faint pink band in lanes 1-4that contain ssDNA and dsDNA. The ncPNA/target polynucleotide hybrid wasexpected to migrate more slowly than either the single-stranded ordouble-stranded DNA 40 bp standards in lanes 1-4. Indeed, at theexpected location was a region that lacked color after photoactivation.These “holes” show up as a darker area on the second panel of FIG. 7identified the presence of the P/TP hybrid.

The method of identifying the target polynucleotides in gel-basedsystems according to the methods disclosed herein can be adapted to anygel-based method of identifying target polynucleotides, includingSouthern, Northern, and Northwestern blotting techniques. Additionally,other agarose gels containing the dye 3,3′-diallylthiacarbocyanineiodide dye demonstrated similar results. Other dyes are anticipated towork similarly

Example 7

This example demonstrates detection of single nucleotide polymorphisms(SNPs) using two nucleic acid analog sequences.

An example of the strategy for detecting point, insertion, and deletionmutations can be found in FIGS. 6C and 6D. PNAs which only partiallyhybridize to target DNA (due to mutations) may form helical duplexes tooshort to participate in light-activated photobleaching of DiSC₂(3). Thefollowing experiment illustrates the affects of point mutations in theassay for the detection of SNPs.

The sequence of ncPNA1 was 5′ Bio-OOOOO-GATAGTGGGATTGTGCGT 3′ [SEQ IDNO:1]. The sequence of ncPNA2 was 5′ TCACATCAATCCACT-lys 3′ [SEQ IDNO:21]. The “0” represents the linker molecule 8-amino-3,6-dioxaoctanoicacid.

Table 5 lists the DNA oligonucleotide sequences with the mismatch basesin lower case. 100 μM single-stranded DNA stock solutions containingdifferent single-stranded DNA 12-mers were prepared. Wild-type DNAoligonucleotide [considered to be the (−) strand] was fullycomplementary to six bases on ncPNA1 (3′ end) and to six bases on ncPNA2(5′ end). Four other DNA oligonucleotides contained 1 or 2 or 4 pointmutations (mu1, mu2, mu3, mu4) were mismatches with bases on eitherncPNA1 or ncPNA2. Each of the 5 DNA oligonucleotides also had an exactcomplementary DNA oligonucleotide [considered to be the (+) strands].

To prepare the double-stranded complementary and mutated sequences, 1.2μl of 100 μM of each (+) DNA strand was mixed with 1 μl of the 100 μMcomplementary (−) DNA strand and diluted to a final concentration of 2μM. These solutions were heated to 95° C. for 5 minutes and allowed tocool to room temperature to promote annealing. The sequences of thecomplementary sequence and the mutated sequences (mu1-mu4) are set forthin Table 5.

TABLE 5 Name + seq 5′ to 3′ − seq 5′ to 3′ wild type GTGCGTTCACATATGTGAACGCAC [SEQ ID NO: 22] [SEQ ID NO: 23] mu1 cTGCGTTCACATATGTGAACGCAg [SEQ ID NO: 24] [SEQ ID NO: 25] mu2 GTGCGTTCACAatTGTGAACGCAC [SEQ ID NO: 26] [SEQ ID NO: 27] mu3 caGCGTTCACATATGTGAACGCtg [SEQ ID NO: 28] [SEQ ID NO: 29] mu4 caGCGTTCAGgtacCTGAACGCtg [SEQ ID NO: 30] [SEQ ID NO: 31]

A ncPNA master mix was made by combining 105 μl of 2 μM ncPNA1, 105 μlof 2 μM ncPNA2, 693 μl of 5 mM phosphate buffer (pH 5.5) containing0.05% Tween® 80, 14% methanol, and 42 μl of 0.75 mM3,3′-diethylthiacarbocyanine iodide dye. This mixture contains finalconcentrations of 220 nM ncPNA1, 220 nM ncPNA2, and 33 μM3,3′-diethylthiacarbocyanine iodide dye. For reactions that containedboth ncPNAs and the dsDNA, 15 μl of each double-stranded polynucleotideand 135 μl of the ncPNA master mix was added to each tube. From eachtube a 50 μl aliquot of each mix was transferred to three wells of thewhite microtiter plate (Greiner).

A control “DNA and dye” only tube that contained complementary DNAoligonucleotides and dye was prepared by combining 20 μl of annealedwild-type DNA, 8 μl of a 0.75 mM solution of3,3′-diethylthiacarbocyanine iodide dye, and 172 μl of 5 mM phosphatebuffer (pH 5.5) containing 0.05% Tween® 80 and 14% methanol. Three 50 μlaliquots of this mixture was dispensed into three wells. Two control“PNA and dye” tubes containing ncPNA1 or ncPNA2 were made by combining20 μl of either ncPNA1 or ncPNA2, 8 μl of the 0.75 mM solution of3,3′-diethylthiacarbocyanine iodide dye, and 172 μl of the 5 mMphosphate buffer (pH 5.5) containing 0.05% Tween® 80 and 14% methanol.50 μl of this mixture was aliquoted into three wells. The plate wasplaced in the Tecan Genios microplate reader and an initial fluorescencewas read. Samples were exposed to the Aurora 50/50 fluorescent light andreadings were taken after every one minute of light stimulus for fiveminutes.

As can be seen in the FIG. 8, fluorescence emission over a period offive minutes remained substantially stable at about 27000 to 32000relative fluorescence units (RFUs) in sample mixtures lacking ncPNA1 orncPNA2 or exactly complementary DNA relative to the nucleic acid analogsequence defined by both ncPNA1 and ncPNA2. After four minutes of lightexposure, the fully complementary sequence can easily be differentiated(open diamonds) from the ncPNA only (1 or 2), DNA only and SNP DNA withboth ncPNAs by the substantial reduction in fluorescence. Interestingly,the start of fluorescence emission reduction started in samples thatincluded the SNP variances mu1 and mu2 at about four to five minutes ofincubation. This observation is extended in the data illustrated in FIG.9, when the fluorescent emission after light stimulation of the mixturecontaining a fully complementary sequence versus a mixture containing amutated oligonucleotide with two or four mutations (i.e., one or two, 2base-pair changes). Again, where one of the two nucleic acid analogs(ncPNA1 or ncPNA2) were absent, the fluorescence emission remainedunwaveringly high at about 32000 relative fluorescence units. Lackingexactly complementary DNA with respect to the sequence of ncPNA1 andncPNA2 provided the same result, essentially. Where both nucleic acidanalog and exactly complementary DNA is included in the mixtures,fluorescent emissions dropped off dramatically at some point after thesecond minute of light exposure, and decidedly so between the third andfourth minute of exposure. The reactions using the same ncPNAs withvariants having two or four mismatches, fluorescent emission could beseen to have started dropping off by the fourth minute of lightexposure.

Example 8

This example compares the two detergents, Tween® 80 and Tergitol® NP-40,in a buffer containing 5 mM sodium phosphate (pH 5.5) in reactions withand without P/TP hybrids to demonstrate increased dye resistance tophotoactivation.

In the following example, identical mixtures were made in buffercontaining either 0.05% Tergol® NP-40 (Sigma Catalog NO: 127087) or0.05% Tween® 80 (Sigma, Catalog NO: P1754). The use of the white platecorrelated with readings of an accelerated reaction rate

A 100 μM stock solution of ncPNA having the sequence 5′Bio-OOOOO-GATAGTGGGATTGTGCGT 3′ [SEQ ID NO: 1] was diluted to 2 μM in 5mM sodium phosphate buffer (pH 5.5) with 0.05% Tween® 80 (“tw80buffer”). An identical dilution was created using 5 mM sodium phosphatebuffer (pH 5.5) with 0.05% NP-40 (NP40 buffer). In separate tubes, a 100μM stock of complementary DNA with the sequence 5′ ACGCACAATCCCACTATC 3′[SEQ ID NO:20] was diluted to a concentration of 2 μM using either thetw80 buffer or the NP40 buffer as above.

The mixture set forth in Table 6 was made.

TABLE 6 Component amount multiplier Total ncPNA 5 μl × 13 65 μl Oligo 5μl × 13 65 μl Buffer (tw80) or (NP40) 38 μl  × 26 988 μl  Dye (0.75 mM)2 μl × 26 56 μlThe dye was first added to the buffer and mixed. For the P/TP reactions,520 μl of buffer/dye mixture, 65 μl of ncPNA, and 65 μl of oligo wereadded and mixed. For the “Dye Only” control, 520 μl was transferred to afresh tube and an additional 130 μl of buffer (either with Tw80 or NP40)was added. Aliquots of 50 μl were dispensed into 12 wells (for eachdetergent buffer and the “Dye Only” control) of a white Greiner 96-wellmicrotiter plate (Catalog NO: 655088).

The plate was then placed in a Tecan Genios microplate reader and aninitial fluorescence was measured. The plate was exposed to the Aurora50/50 photoactivator and readings were taken after every 1 minute oflight exposure up to 7 minutes of total exposure.

The fluorescence at various times (of total exposure to the Aurora 50/50light) is presented graphically in FIG. 10. The lines on the graphrepresented data from the identified reactions: Solid circle, thecontrol of dye only in tw80 buffer; solid triangles, the ncPNA/oligoreaction in tw80 buffer; the solid squares, the control of dye only inNP40 buffer; and solid diamonds, the ncPNA/oligo reaction in NP40buffer. As clearly indicated on the graph, the two controls of dye onlydemonstrate that the fluorescent emissions remained constant over theobserved seven minutes of light exposure. Additionally, dye in solutionwith Tween® 80 had and maintained fluorescent emission at approximatelya 50% greater level than that of the dye in solution with NP-40. Bothreactions demonstrated observable responses by the second minute afterlight exposure began, and dramatically so by the third minute after thestart of light exposure.

Accordingly, this example demonstrates two aspects of the presentinvention: (1) use of surfactants NP-40 and Tween® 80, both non-ionic,correlated with increased dye fluorescence stability uponphotoactivation, and (2) Tween® 80 correlates with about a 50% increasedlevel of fluorescent emission as compared to reactions with NP-40.

Example 9

This example illustrates the effect that different plates have regardingthe profiles of the reactions of the amino acid analog, targetpolynucleotide, and dye, in accordance with the present invention.

The microliter plates listed in Table 7 were obtained and tested todetermine whether different color schemes of such materials have aneffect on the reaction of a nucleic acid analog, a complementary targetpolynucleotide, and a dye, as provided by the present invention.

TABLE 7 Plate Brands Description Color Scheme 1. Greiner 655073 96-wellwhite with white bottom 2. Greiner 655088 96-well, μclear black withclear bottom 3. Greiner 355892 96-well, glass bottom black with clearbottom 4. Greiner 655095 96-well, μclear white with clear bottom 5.Greiner 655096 96-well, μclear black with clear bottom 6. Greiner 781091384-well black with clear bottom 7. Greiner 788096 384-well, smallvolume black with clear bottom 8. Greiner 788092 384-well, small volumeblack with clear bottom 9. Greiner 781892 384-well, glass bottom blackwith clear bottom 10. NUNC 436014 96-well, streptavidin clear with clearbottom 11. NUNC 265302 96-well white with white bottom 12. NUNC 23710596-well black with black bottom 13. NUNC 265301 96-well, optical bottomblack with clear bottom 14. NUNC 265301 96-well, optical bottom whitewith clear bottom 15. Costar 3601 96-well, high binding black with clearbottom 16. Corning 3651 96-well black with clear bottom 17. Costar 363196-well black with clear bottom 18. Costar 3615 96-well, special opticsblack with clear bottom 19. Costar 3632 96-well white with clear bottom20. Costar 3693 96-well, ½ area white with white bottom 21. BD 96-well,streptavidin black with black bottom Biosciences 353241 22. BD 96 wellwhite with white bottom Biosciences 354742 23. Matriplate 384-well,glass bottom black with clear bottom

Each plate listed in Table 7 was tested. In a 50 μL test reaction, an18-mer ncPNA with the sequence 5′ GATAGTGGGATTGTGCGT 3′ [SEQ ID NO: 1](N-terminus to C-terminus) and a complementary DNA oligonucleotidetarget (final concentration 200 nM for both) were mixed with3,3′-diethylthiacarbocyanine iodide dye (final concentration 300 μM) ina phosphate buffer with NP-40. A 50 μL control reaction mixture ofphosphate buffer with NP-40 with 3,3′-diethylthiacarbocyanine iodide dye(final concentration) was run simultaneously. Light was applied to theplates, and the fluorescence intensity over time was measured.

In plates with clear bottoms, light was projected upwards through thewell. The distance between the light source and the bottom of the wellwas approximately ¼ inch. In plates with black or white bottoms, lightwas projected downwards into the well at a distance of about 1 inch fromthe surface of the liquid. A fluorescent reading prior to light exposurewas taken followed by subsequent readings every 60 seconds duringexposure to light. Data were plotted as fluorescence vs. time with eachdata point representing at least 12 identical reactions, plus and minusthe standard deviation.

FIGS. 11A-D present the data graphically of replicate test reactions andcontrols reactions which were run in a white plate with white bottom(FIG. 11A); a white plate with clean bottom (FIG. 11B); a black bottom(FIG. 11C); and a black plate with clear bottom (FIG. 11D). Thisexperiment demonstrates a considerable effect of the reflective (i.e.,white) or absorptive (i.e., black) nature of the plate on the rate offluorescence decay of 3,3′-diethylthiacarbocyanine iodide dye.

When the light source projected downward into the reaction (FIGS. 11Aand 11B), there was an accelerated rate of fluorescence decay of thetest reaction while the control reaction demonstrated minimalfluorescence decay. When a white plate with white bottom was used, thefluorescence of the “Dye Only” reaction increases from time zero to 15minutes (FIG. 11B). In a black plate with a black bottom the rate offluorescence decay of the test reaction was significantly slower thanthe same reaction in a white plate with the white bottom.

In plates containing clear bottoms (where the light was projectedupwards through the wells), the white plates demonstrated a faster rateof fluorescence decay of the test reactions compared to the black plates(FIGS. 11C and 11D), reaching the minimum observed value in about threeminutes versus about seven minutes. The control reactions alsodemonstrated a slightly faster rate of fluorescence decay in the whiteplates compared to the black plates.

In general, the results were not significantly different using differentbrands (i.e., NUNC, Greiner, or Costar) of microplates. It was observed,however, that the black plates correlated with nearly a 50% increase influorescence emission relative to the white plates (see the 0 timefluorescence level for each set of experiments, for example). It wasalso observed that the white plates correlated with a test reaction thatwas faster in reaching the minimum fluorescence level.

Example 10

This example describes an experiment and results thereof that tested theuse of a modified reaction buffer that included surfactant and alcohol.

A modified phosphate buffer was identified that provides greatlyenhanced stabilization of dye and reduced background signal. Themodified buffer consisted of the 5 mM phosphate buffer described above,plus 0.05% Tween 80 and 14% methanol. Stabilization of the dye was alsoenhanced by using less light. The reactions described in this examplewere conducted in a white 96 well flat white bottom microtiter plate(Greiner).

Reactions were set up in triplicate and loaded on the microtiter plate.Reaction mixes were made from 7.5 mM 3,3′-diethylthiacarbocyanine iodidedye stock, 100 μM ncPNA stock, Bio 18 (5′ Bio-ooooo-GATAGTGGGATTGTGCGT3′ [SEQ ID NO:1]), 100 μM complementary DNA set (5′ GATAGTGGGATTGTGCGT3′ [SEQ ID NO:1]; 5′ ACGCACAATCCCACTATC 3′ [SEQ ID NO:20]), 1 μl of eachhomologous DNA pair was mixed equally in annealing buffer (Sambrook, etal) at a concentration of 2 μM and heated to 95° C. for five minutes,then allowed to cool to room temperature. The ncPNA were diluted to 2 μMworking stock.

In a PCR strip tube, 20 μl of 2 μM ncPNA and 20 μl of complementary DNAwas mixed, making a test reaction mixture. 20 μl of ncPNA and 20 ofbuffer was added to a well making a ncPNA/dye control reaction mixture.20 μl of DNA and 20 μl of buffer were added to a well making a DNA/dyecontrol reaction mixture, and 40 μl of buffer was added to the dye onlycontrol reaction mixture. A master dye buffer mix was made by adding 36μl of 0.75 mM dye to 684 μl of 5 mM PO₄ buffer plus 0.05% Tween® 80 with14% methanol. The solution was mixed and 160 μl was dispensed to each ofthe 4 wells of the PCR strip tube. The solutions were mixed and 50 μl ofeach reaction mix was dispensed into 3 wells (triplicate) of the whiteplate. The plate was placed in the Tecan Genios microplate reader and aninitial fluorescence measurement was taken (before photoactivation).Samples were exposed to the Aurora 50/50 fluorescent light at half itslight intensity and fluorescence measurements were taken after every 1minute of light exposure for 10 minutes.

The results are graphically displayed in FIG. 12. The dye only controlreaction mixture (▬) and ncPNA/dye control reaction mixture (x)exhibited greatly enhanced stabilization of the zero time fluorescentemission (referred to here as the background signal) by showingvirtually no reduction in fluorescence. The DNA/dye control reactionmixture showed a small reduction in fluorescence, however the reductionwas negligible through at least seven minutes of light exposure. Thetest reaction mixture had a fast rate of decrease in fluorescentemission, starting within the third minute. The addition of the alcoholin tandem with surfactant correlates with the enhanced dyestabilization; as illustrated by negative controls in FIG. 11, forexample.

Example 11

This example illustrates a fast method for preparing a cellular samplefor testing whether a particular target polynucleotide is present.Reaction buffer (0.5 mM phosphate) containing 0.05% NP-40 was used topermeabilize and/or lyse fresh overnight cultures of E. coli and B.cereus grown in tryptic soy broth. Tests from the diluted samples wereprepared by using 300 μl from each culture sample. Samples were spundown to a pellet and supernatant removed. Pellets were resuspended in390 μl of reaction buffer and incubated at room temperature for 10minutes before use in the test reaction. Each test sample contained 10pmoles of 16S nucleic acid analog (ncPNA) probe (5′-ACT GCT GCC TCC CGTAG-3′ [SEQ ID NO:8]) or 10 pmoles of HCV-specific biotinylated nucleicacid analog (ncPNA) probe (5′ Bio-(O)₁₀-CGCAGACCACTA 3′ [SEQ ID NO:35]),5 μl of each lysate dilution test and 4 nmoles of3,3′-diethylthiacarbocyanine iodide (DTCC dye). The so-constituted testsamples were diluted to a 50 μl total reaction volume with 5 mM tw80buffer. Tests were done either with 16S ncPNA as probe, which shoulddetect the E. coli and B. cereus bacteria, and a non-specific HCV probethat should not recognize any bacterial DNA in this system. The positivecontrol well contained 16S ncPNA probe, 10 ng of isolated E. coli DNA,dye and buffer. The probe only well contained either the HCV ncPNA probeor 16S ncPNA probe, and dye and buffer. The dye only well contained dyeand buffer.

ncPNAs were reconstituted in DNase-, RNase-free water to 100 μM stockand further diluted to 2 μM working stocks. The DTCC dye was dissolvedin DMSO to 8 mM stocks. This was further diluted to 2 mM working stockin 5 mM phosphate buffer (pH 5.5)+NP-40 0.05%. An initial fluorescencereading was taken at time zero in the Tecan Genios microplate readerwith the wavelengths set at 535 nm for excitation and 590 nm foremission. The samples were then exposed to a light stimulus using theAurora 50/50 for 1 minute intervals with fluorescence reading beingtaken after each exposure for 10 minutes.

The data were used to calculate the percent change in the test wellscontaining bacterial lysate and either the HCV ncPNA probe or the 16SncPNA probe compared to the reaction well containing dye and theanalogous HCV ncPNA probe or 16S ncPNA probe. By so comparing thepercent change in fluorescence readings after differing numbers ofminutes of light exposure, the rate of change of fluorescence emissionwas determined (which is directly related to the presence of targetmolecules).

The results are graphically presented in FIG. 13. A strong signal can beseen from the positive control reaction containing bacterial DNA. Testsamples containing the 16S ncPNA probe with either E. coli or B. cereuslysates produced robust signals as well, which demonstrates that themethod of the present invention is well-suited for bacterialidentification using suitable sequences of nucleic acid analogs. Testsamples with bacterial lysate and the non-specific HCV ncPNA proberemained at background levels, indicating that one should not expectfalse-positive results from the herein described method despite the useof crude bacterial lysates for the samples that were tested.Accordingly, the permibilization/lysis buffer producing a crude lysateis sufficient for the detection method disclosed herein.

Example 12

This example further explores using a crude bacterial lysate as thesource of a sample for testing whether a target polynucleotide ispresent.

Reaction buffer (0.5 mM PO₄) containing 0.05% NP 40 was used topermeabilize and/or lyse diluted aliquots of fresh overnight cultures ofE. coli cells that were grown in tryptic soy broth. A sample from theculture was serial diluted and plated on LB agar plates for later colonycounts to determine the cell concentration. Analogous serial dilutionsof E. coli cells (10 fold dilutions) were made in tryptic soy broth. A“zero” sample contained broth only. Samples were prepared by taking 250μl of each diluted sample, centrifuging to pellet the cells, andremoving supernatant. Pellets were resuspended in 390 μl of buffer andincubated at room temperature for 10 minutes before an aliquot thereofwas used in the test reaction. Each test sample contained 10 pmoles of16S ncPNA probe (5′ ACT GCT GCC TCC CGT AG 3′ [SEQ ID NO:8]), 5 μl ofeach lysate dilution, and 4 nmoles of 3,3′-diethylthiacarbocyanineiodide (DTCC dye); placed in a 50 μl total reaction volume using 5 mMPO₄/NP-40. The positive control well contained 16S ncPNA probe,complementary oligo, dye and buffer. The probe only well containedprobe, dye and buffer. The dye only well contained dye and buffer.

ncPNAs were reconstituted in DNase- and RNase-free water to 100 μM stockand further diluted to 2 μM working stocks. The DTCC dye was dissolvedin DMSO to 8 mM stocks. This was further diluted to 2 mM working stockin 5 mM phosphate buffer (pH 5.5)+0.05% NP 40. An initial fluorescencereading was taken at time zero in the Tecan Genios microplate readerwith the wavelengths set at 535 nm for excitation and 590 nm foremission. The samples were then exposed to a light stimulus using theAurora 50/50 for 2 minute intervals with fluorescence reading beingtaken after each exposure for 20 minutes.

The data were used to calculate the percent change in the reaction testwell containing the ncPNA probe and the diluted bacterial lysatecompared to the reaction well containing only the ncPNA probe/dye.

FIG. 14 graphically displays the results. The samples of bacteria thatwere tested contained the following numbers of bacterial cells: 4.3million (Δ); 430,000 (▬); 43,000 (*); 4,300 (|); 430 (♦); 43 (◯); and 0(□). Additionally a positive control (▪) was included, which had a verystrong signal. Similarly, the signal from a reaction mixture having4,300,000 cells was also very clear, and the signal from a reactionmixture using 430,000 cells, while still clear, departed from therobustness of the signal from a positive control reaction mixture.Counts of 43,000 cells and below resulted in data that was virtuallyindistinguishable from background levels. These results demonstrate theability of the method to quantitate in a system of crude cellularlysates.

Example 13

This example illustrates further the combined effect of Tween 80 andmethanol in the reaction buffer, and compares the effect to that ofmethanol alone.

The effect of methanol in conjunction with Tween 80 at greatlyincreasing the signal-to-noise ratio in a reaction was described above.(See Example 10). The combined effects with Tween 80 are much greaterthan when the same concentration of methanol is used alone. Tworeactions were made, one with Tween 80 and another without. The modifiedbuffers were made having the following content: (1) 5 mM PO₄ buffer plus0.05% Tween 80 with 14% methanol; and (2) 5 mM PO₄ buffer with just 14%methanol. Reactions were run using a white 96-well flat-bottommicrotiter plate (Greiner) and the Aurora 50/50 fluorescent light athalf its intensity.

Reactions were set up in triplicate and loaded in the wells of the96-well microtiter plate. Reaction mixes were made from the following:7.5 mM Dye stock, 100 μM ncPNA stock, (5′ Bio-ooooo-GATAGTGGGATTGTGCGT3′ [SEQ ID NO:1]), 100 μM complementary oligo set (5′ GATAGTGGGATTGTGCGT3′ [SEQ ID NO: 1], 5′ ACGCACAATCCCACTATC 3′ [SEQ ID NO:20]); 1 μl ofeach exactly complementary oligo pair was mixed equally in annealingbuffer (Sambrook, et al) at a concentration of 2 μM and heated to 95° C.for 5 minutes, then cooled to room temperature. The ncPNA were dilutedto 2 μM working stock.

A master dye buffer mix was made by adding 36 μl of 0.75 mM dye to 684μl of 5 mM PO₄ buffer plus 0.05% Tween® 80 with 14% methanol. Thesolution was mixed and 160 μl was dispensed to each of 4 wells of astrip tube (Perkin Elmer, Catalog #N801-0580). In the first tube, 20 μlof 2 μM ncPNA and 20 μl of DNA were mixed, making a test reactionmixture. Additionally, these different negative controls were tested inparallel: in a second tube 20 μl of ncPNA and 20 μl of buffer was addedmaking a ncPNA/dye only reaction mixture, in a third tube 20 μl of oligoand 20 μl of buffer was added making the oligo/dye only reactionmixture, and to a forth tube 40 μl of buffer only was added making thedye only reaction mixture. An identical set of reaction mixtures wasmade as above, but 5 mM PO₄ buffer plus 14% methanol (without Tween® 80]was used. The solution was mixed and 50 μl of each reaction mix wasdispensed into 4 wells of the white plate. The plate was placed in theTecan Genios microplate reader and an initial fluorescence was readwithout light exposure. Samples were exposed to the Aurora 50/50fluorescent light at half its light intensity, as above, and readingswere taken after every minute of light exposure for 10 minutes. The datawere recorded as presented graphically in FIG. 15.

Fluorescent emission at T₀ was greater for reaction mixtures containingthe surfactant/alcohol combination in the reaction buffer as compared toreaction mixtures not containing the surfactant/alcohol combination inthe reaction buffer. Moreover, all negative control reaction mixtures(PNA+dye, ▪; DNA+dye, ●; and dye only, x) showed increased stability ofthe fluorescence signal over time, which translates to reducedbackground noise compared to target polynucleotide, when methanol isused in conjunction with Tween® 80 (left graph set). The dye only andncPNA/dye reaction mixtures exhibited very little change in fluorescencesignal over time. The DNA/dye reaction mixture exhibited minor change influorescence signal over time while the test reaction mixture exhibiteda very rapid reduction in fluorescence signal over time.

When 14% methanol is used alone there is a much lower relative change influorescence signal of the test reaction mixture as compared to thechange in fluorescence signal of all control reaction mixtures.

Example 14

This example illustrates two methods for analyzing a change in anoptical property as a function of time as it relates to the presentinvention.

Experimental data were obtained using the following protocol. Samplescontaining or not containing genomic nucleic acid (NA) isolated fromMycobacterium tuberculosis (MTB CDC 1551) were prepared and tested usinga protocol of the present invention. Accordingly, the samples were usedin generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.08 ng/μL in molecularbiology grade water (Hyclone, catalog #SH30538.03).

At this point, a 50 μM PNA probe mix was prepared from freezer stocks.Sequences used were Sequence ID NO: 42, 43, 44, 45, 46 and 47. Freezerstocks (at ˜200 μM in H₂O) were put in a hotblock at 65° C. for fiveminutes, and a 50 μM solution of each ncPNA was prepared by diluting thefreezer stock ncPNA in molecular biology grade H₂O (Hyclone, catalog#SH30538.03). Equal volumes of each 50 μM ncPNA solution were added tocreate a 50 μM total PNA concentration, i.e., 8.33 μM per ncPNA. This 50μM PNA probe mix was put in the hotblock at 65° C. for five minutes, andwas then further diluted in phosphate buffer (10 mM) with surfactant toa final concentration of 320 nM. The 320 nM PNA probe mix was placed ina water bath at 65° C. for five minutes, removed, and given 30 minutesto cool to room temperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) intophosphate buffer with surfactant to a working concentration of 36 μM.

Serial 1:2 dilutions of the 0.08 ng/μL MTB NA (in water) were prepareddown to a low concentration of 0.0025 ng/μL; referred to herein as “DNAstandards”. Aliquots of 25 μL of each of the DNA standards weredispensed into a 384 well white/clear plate (NUNC, catalog #242763) toset up a concentration curve with six replicates in individual columnson the plate. The columns were set up as follows: Rows 1-6 and 9-14 wereassay wells with 2 ng, 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, and 0.0625 ngMTB DNA per well, Row 7 and 15 were control wells with 0 ng DNA and Row8 and 16 were control wells with 2 ng MTB DNA and no PNA.

Because of the sensitivity of the optical measurements, care was takento ensure that the surfaces of the solutions in the microtiter platewere uniform. The solutions were dispensed using a reverse-pipettingtechnique described by B. Brando et al. (CYTOMETRY 42:327 (2000)).Briefly, the technique involves pushing the plunger on a mechanicalpipettor past the first stop for the initial reagent draw, and pushingthe plunger only to the first stop for dispensing, ensuring that a smallvolume of liquid remains in the pipettor after dispensing. The assaymicroplate was briefly centrifuged at 500 RPM (34×g) in a Sorvallbenchtop centrifuge (Model RT6000D) after the addition of each reagenttype.

After the DNA standards were dispensed into the microplate, and themicroplate was centrifuged, two dye mixtures were then prepared. Thereaction set-up steps involving the dye were performed in a dimly litroom. The first control dye solution was prepared by mixing equalvolumes of 36 μM DiSC₂(3) with phosphate buffer (with surfactant). This18 μM DiSC₂(3) control solution was mixed by inversion in a 15 mLconical tube, and poured into a reagent reservoir. A 25 μL aliquot ofthe control dye solution was then dispensed to each well along Row 8 [H]of the microplate. The second, dye+PNA probe mix was prepared by mixingequal volumes of 36 μM DiSC₂(3) with the 320 nM PNA probe mix. This 18μM DiSC₂(3)+160 nM PNA probe mix was mixed by inversion in a 15 mLconical tube, and poured into a reagent reservoir. A 25 μL aliquot ofthe dye+PNA probe mix was then dispensed to each well along Rows 1-7[A-G] of the microplate. The microplate was then briefly centrifuged at500 RPM.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 44 minutes totalexposure to light.

Dye bleaching reactions catalyzed by a PNA-DNA complex in the presenceof the above light stimulus exhibit decreasing absorbance as a functionof time when monitored at the absorption maxima of the dye, typically556 nm. Typical absorbance vs. time data for the varying amounts of MTBDNA as described in the preceding experimental protocol are displayed inFIG. 18. Each data point on the graph is the average of 48 individualdeterminations on the 384 well assay plate, standard deviations are notshown in the graph but were typically less than 5%.

Absorbance change vs. time data can be used to estimate concentration oftarget nucleic acids in two ways. In the first preferred methodabsorbance rate changes are extracted from the experimental data duringthe initial portion of the reactions when the rate of change varieslinearly with time (FIG. 19). Absorbance changes in this linear regionmay be approximated by a linear equation in the first 2-10 min ofobservation, after this initial phase the reactions exhibit significantnonlinearity. The initial data can be fit to a linear equation using themethod of least squares and the slope of the initial data can then beestimated from the resulting linear equations. This method has theadvantage of also allowing the correlation coefficient R² to becalculated for the data thereby providing a measure of how good the datafit to the linear equation. Alternately the initial slope of a reactionmay be estimated by subtracting the absorbance at a fixed time point,say 10 minutes, in the linear portion of the reaction from the startingabsorbance at 0 minutes and dividing the result by the time intervalbetween the two points. Initial rates of change for differentconcentrations of MTB DNA were calculated for the data displayed in FIG.19 by fitting the data to a linear equation using the “add trendline”analysis function in the program Microsoft Excel. The initial slopeswere also calculated subtracting absorbance at time 0 from absorbance at10 minutes and dividing the result by 10. The results of both methods ofcalculation are shown in Table 8 and are in good agreement with oneanother.

TABLE 8 Estimation of Initial Reaction Rate Slope Amount MTB 2.0 ng 1.0ng 0.50 ng 0.25 ng 0.125 ng 0.0625 ng 0 Average 0.4853 0.4895 0.49350.4912 0.4958 0.4966 0.4914 Absorbance 0 min Average 0.2370 0.33400.3935 0.4192 0.4311 0.4360 0.4340 Absorbance 10 min Slope 2.48E−021.56E−02 1.00E−02 7.20E−03 6.47E−03 6.06E−03 5.74E−03 0-10 min Slopefrom 2.48E−02 1.56E−02 1.01E−02 7.37E−02 6.66E−03 6.26E−03 5.96E−03Graph Fit Line R² 0.994 0.997 0.999 0.997 0.995 0.994 0.992 Value

In addition, R² values for the fitted lines indicate that the data fitwell to a linear equation. Slope values are expressed as positivenumbers. A dose response curve constructed from these data exhibits alinear relationship between analyte concentration and initial slopevalue as shown in FIG. 20. Linear fitting of the experimental data wasaccomplished using “add trendline” analysis function in the programMicrosoft Excel. Once again examination of the R² value for this datashow they fit well to a linear equation. Thus the assay method whencombined with this process of data reduction allows quantitativemeasurements of target analytes in a straightforward manner.

A second method of data reduction can also be used to analyze dyebleaching reactions in order to derive dose response curves. In thismethod the time required to observe a given change in absorbance isestimated for each analyte concentration and that time is plotted vs.analyte concentration to produce a dose response curve. The presentexperiments start at an absorbance of approximately 0.5 absorbance unitsand the time interval for each reaction to reach 0.3 absorbance unitswas estimated graphically from the data as shown in FIG. 21. Reductionby 0.2 absorbance units has a physical sense: this change in absorbancecorresponds to a reduction of transmission of approximately 1.6 fold(log(1.6)=0.2). The relation between time to absorbance and analyteconcentration derived by this method is not typically linear, FIG. 22.The method does provide a good way of comparing many experimentalconditions, especially if the data is not well behaved with the InitialSlope Method.

Example 15

This example sets forth tests conducted to assess the effects ofdifferent detergents on diagnostic reactions of the present invention.

In a 96-well white with clear bottom NUNC microtiter plate, 100 μL of a5 mM phosphate buffer (pH 5.5) was mixed with one of a collection ofdetergents (also referred to as surfactants), which are listed in Table9.

TABLE 9 Trade or Common Name of Type of Detergent Studied* Synonyms;Molecular Formula Detergent CHAPS (100 mg) 3-[(3- zwitterioniccholamidopropyl)dimethylammonio]- 1-propanesulfonate hydrateoctyl-β-glucoside (100 mg) octylglucopyranoside nonionicoctyl-β-D-thioglucoside (100 mg) octylthioglucopyranoside; OTG nonionicSurfact-Amps ™ X-100 (a 10% octylphenol (ethoxylate)_(n) where n isnonionic aqueous solution of Triton ® X- 9 or 10 on average 100)Surfact-Amps ™ X-114 (a 10% octylphenol (ethoxylate)_(n) where n isnonionic aqueous solution of Triton ® X- 7 or 8 on average 114)Surfact-Amps ™ NP-40 (a 10% [octylphenoxy]polyethoxyethanol nonionicaqueous solution of Nonidet P- 40) Surfact-Amps ™ 20 (a 10%polyoxyethylene sorbitan nonionic aqueous solution of Tween ® 20)monolaurate; polysorbate 20; C₅₈H₁₁₄O₂₆ Surfact-Amps ™ 80 (a 10%polyoxyethylenesorbitan monooleate; nonionic aqueous solution of Tween ®80) polysorbate 80 Surfact-Amps ™ 35 (a 10% polyoxyethylene monolaurylether; n nonionic aqueous solution of Brij ® 35) ca. 23 Surfact-Amps ™58 (a 10% polyethylene oxide hexadecyl ether nonionic aqueous solutionof Brij ® 58) *The full collection of detergents studied was purchasedfrom Pierce Biotechnology, Inc., Rockford, IL; catalog #28340.

Each of the “Surfact-Amps” detergents was further diluted with 5 mMphosphate buffer (pH 5.5) to a final concentration of 0.05%. The 100 mgquantities of CHAPS, octyl-β-D-glucoside, and octyl-β-D-thioglucosidewere respectively dissolved in 5 mM phosphate buffer (pH 5.5) to a finalconcentration of 0.05 mg/mL (w/v). Also included with thephosphate/detergent solution was the dye dipropylthiacarbocyanine iodide(DiSC₃(3)), which was included at a final concentration of 18 μM. Acomparison was made between those phosphate/detergent solutions with 100nM (final concentration) of a pre-annealed PNA:DNA hybrid to those samephosphate/detergent solutions without the PNA:DNA hybrid. The PNA:DNAhybrid used was formed by the combination of PNA sequencebiotin-(O)-GATAGTGGGATTGTGCGT [SEQ ID NO: 1] and its complementary DNAoligonucleotide sequence 5′ ACGCACAATCCCACTATC 3′ [SEQ ID NO:20]. Alsoincluded in the study were phosphate solutions with no detergent, whichwere with or without the PNA:DNA hybrid. The Aurora 50/50 light was usedfor photoactivation, with fluorescence readings (excitation 540 nm,emission 585 nm) taken at T₀ and 2 minute intervals out to 20 minutesusing a dual-monochromator, multi-detection microplate reader known asSpectramax M5 (sold by Molecular Devices Corporation, Sunnyvale,Calif.). The Spectramax M5 microplate reader generated data points inrelative fluorescence units (RFUs), which were recorded and presented inthe tables below.

Table 10 presents the fluorescence data from light-exposed reactionmixtures that included 18 μM DiSC₃(3) with 0.1 μM PNA:DNA in 5 mMphosphate buffer with different detergents at 0.05% concentration.

TABLE 10 Time Relative Fluorescence Units (RFUs) at Stated Minutes ofLight Exposure exposed to octyl-B- light (min) CHAPS glucoside1-s-B-thioglucopyranoside Tween20 Tween80 NP40 TritonX100 TritonX114Brij 35 Brij 58 H₂O 0 3171 2949 3106 8623 9147 12095 10841 7876 85649364 3036 2 2153 2106 2237 7283 7737 10242 9407 6706 7394 8154 2112 41275 1384 1593 5583 5923 8324 7785 5147 6023 6621 1585 6 396 746 8293438 3846 6221 6065 3323 4382 4879 1109 8 116 246 337 1376 1643 41784357 1548 2300 3077 732 10 102 106 124 544 690 2190 2830 1097 1206 1368452 12 93 87 103 385 460 1447 1487 928 649 751 308 14 85 77 91 328 3891120 1011 852 483 548 197 16 82 66 82 301 352 972 818 789 418 477 135 1878 62 76 279 322 885 731 731 381 427 94 20 75 59 73 263 306 832 670 699357 403 79

Each detergent gave a different initial T₀ emission reading, but allbehaved similarly in the rates of change in fluorescence uponphotoactivation when the PNA:DNA hybrid was present. Based on the dataset forth above, from T₀ T₈, i.e., the first eight minutes of lightexposure, the rates of change in fluorescence in the reaction mixturewere each between nearly 800 and about 1000 RFU per minute. In contrast,without a detergent included in the reaction mixture, the rate of changeover the same eight minutes was about 285 RFU per minute, i.e., aboutone-third the rate of the detergent-containing reaction mixtures.Because of the approximately three-fold increased rate of changeexhibited by the detergent-containing protocol, one can perceive adifference in optical property with the naked eye in a 50 μl to 100 μlreaction mixture within a minute or two, and certainly within fiveminutes, of the start of photoactivation at ambient temperature.

Table 11 illustrates the percent change between the “ncPNA:DNA” reaction(data in Table 10) and the “dye only” reaction (data in Table 12),relative to the “dye only” reaction at each time point. Comparativeanalysis between the results of reaction mixtures after a given time oflight exposure and having the identified detergents or water in thepresence of the P/TP or not. The percentage difference is shown inaccordance with the following formula:[[(RFU_(Table C))−(RFU_(Table A))]÷RFU_(Table C)]×100.

A remarkable similarity of effect is seen for all tested detergents. Thepresence of and type of detergent in the reaction affects the time atwhich maximal percent change between reactions containing dye only andreactions containing dye with ncPNA:DNA occurs. All reactions withdetergent (at 0.05%) gave a greater percent change than reactionswithout detergent relative to dye only reactions.

TABLE 11 Percent Change Relative to “Dye Only” Time RelativeFluorescence Units (RFUs) at Stated Minutes of Light Exposure exposed tooctyl- light (min) CHAPS B-glucoside 1-s-B-thioglucopyranoside Tween20Tween80 NP40 TritonX100 TritonX114 Brij 35 Brij 58 H₂O 0 3.8 5.2 1.9 0.61.0 0.7 0.4 1.8 0.7 2.5 5.8 2 27.3 23.3 22.4 9.7 9.0 7.4 6.3 5.6 8.6 8.74.8 4 52.1 29.8 24.1 26.6 26.1 20.4 18.0 19.6 20.9 20.9 10.3 6 81.0 53.952.0 50.8 49.5 37.6 33.1 43.7 39.2 38.1 18.7 8 93.9 81.6 77.7 79.5 77.756.6 50.3 71.8 66.4 59.2 27.1 10 93.8 89.5 89.6 91.6 90.4 76.7 66.6 78.582.1 81.6 36.1 12 93.9 89.7 89.8 93.9 93.4 84.3 82.1 80.6 90.1 89.7 37.614 93.7 88.7 88.9 94.7 94.3 87.6 87.5 80.8 92.4 92.2 42.8 16 93.2 87.187.2 94.9 94.7 89.1 89.7 80.8 93.2 93.1 39.5 18 92.8 84.7 85.5 95.2 95.089.8 90.4 80.7 93.7 93.7 35.6 20 92.1 81.4 83.0 95.3 95.2 90.3 91.2 80.793.9 93.9 19.9

Identical experiments containing the same detergents but excluding thePNA:DNA hybrid demonstrated the stabilizing effect that certaindetergents have on the dye, and thus on the optical property of thereaction mixture. The data was generated using 540 nm exposure of thereaction mixture for excitation of the dye and reading of emittedfluorescence at 585 nm. The data generated from reaction mixturescontaining the identified detergents or water without inclusion of aP/TP are set forth in Table 12 below.

TABLE 12 Time Relative Fluorescence Units (RFUs) at Stated Minutes ofLight Exposure exposed to octyl-B- light (min) CHAPS glucoside1-s-B-thioglucopyranoside Tween20 Tween80 NP40 TritonX100 TritonX114Brij 35 Brij 58 H₂O 0 3297 3110 3164 8674 9244 12010 10880 7737 86229600 3222 2 2959 2744 2883 8061 8500 11056 10041 7102 8086 8931 2219 42662 1971 2098 7606 8012 10462 9495 6404 7617 8372 1767 6 2086 1618 17276995 7617 9966 9067 5904 7203 7878 1365 8 1895 1333 1515 6714 7371 96278774 5493 6851 7535 1004 10 1650 1008 1185 6488 7163 9385 8483 5096 67317430 708 12 1518 846 1002 6285 6992 9200 8322 4789 6531 7283 493 14 1350678 817 6133 6853 9065 8090 4432 6311 7058 344 16 1203 513 646 5950 65928890 7918 4120 6190 6921 223 18 1071 409 523 5776 6483 8687 7650 37966021 6750 146 20 960 317 431 5641 6386 8546 7582 3616 5853 6626 98

While a decrease in fluorescence was seen in all of the reactions usingthe various detergents, or no detergent, the fastest rate of change ofthe fluorescence was no greater than about a third of the rate of changenoted above for reactions where the PNA:DNA hybrid was present.Reactions with the two glucoside derivatized detergents, the onlyzwitterionic detergent (i.e., CHAPS), or water behaved similarly: Thesereaction mixtures had T₀ readings that were substantially less thanthose of the other reactions that included Triton® X, Tween®, and NP-40non-ionictype detergents. Furthermore, the low level of fluorescencethat was present in the glucosides or CHAPS degraded further to nearzero over the 20 minute exposure to light. Other dyes were tested,including DiSC₄(3) and DiSC₅(3), and exhibited similar results

Example 16

This example illustrates an embodiment of the present invention where anincreasing fluorescent signal correlates to a decreasing absorbance ofthe reaction mixture, thereby providing a simple method for assessing agiven reaction.

The top of a 96-well microtiter plate (Nunc #265302) was sealed and theoptical bottom was coated with fluorescent yellow paint (Rust-Oleum®,catalog #1942). It is believed that any other optically-identifiedcoating or, in the alternative, an optically-identified inclusion in thematerial of the reaction vessel or in the reaction mixture itself wouldwork equally well. One simple alternative approach to painting of thereaction vessel, for example, includes, for example, affixing tape orplastic to a surface of the reaction vessel.

ncPNA probe and oligonucleotide complement (“oiDNA”) solutions wereprepared by diluting 100 μM stock solutions 1:50 in ddH₂O (Nanopure),resulting in a 2 μM solution of biotinylated ncPNA [SEQ ID NO: 1] and a2 μM solution of the oiDNA that is complementary to the ncPNA [SEQ IDNO:20].

Two master mixes were then prepared: One negative control reactionmixture without target nucleic acid or ncPNA and one test reactionmixture containing the ncPNA and target nucleic acid. The negativecontrol reaction mixture was prepared by adding 24 μL DiSC₂(3) (at 0.75mM in 10% methanol) and 84 μL of 10% methanol to 492 μL reaction buffer(10 mM PO₄+0.05% Tween® 80). The test reaction mixture was prepared byadding 24 μL DiSC₂(3), 60 μL ncPNA, 60 μL oiDNA and 84 μL 10% methanolto 372 μL of buffer. Each mixture was then dispensed to 12 wells of thepainted plate, 50 μL per well.

The fluorescence (excitation at 485 nm, emission at 535 nm,corresponding to the fluorescence of the yellow paint on the plate) ofeach well at zero time (T₀; no light exposure prior thereto) was thenread using a Tecan GENios microplate reader. Average fluorescent signalwas determined for each control or experimental reaction, standarddeviations were calculated, and the data were recorded.

Reactions were then activated with the Aurora 50/50 illuminator for 1minute. Fluorescence was measured with the same parameters as the T₀measurement. This cycle of photoactivation followed by an immediatefluorescence measurement was continued out to 10 minutes of exposure tolight. The data are provided in the following Table 13, wherefluorescence data is presented from test reaction mixtures and negativecontrol reaction mixtures. The fluorescence measured relates tofluorescent material painted on the outside of the reaction vessels.

TABLE 13 RFU* after stated time of light exposure (min) DNA PNA DYE 0 12 3 4 5 6 7 8 9 10 Test + + + 7821 11526 14876 19213 25229 33175 3593836793 37252 37617 37545 Reaction Negative − − + 7748  8397  8928  951310070 10615 11220 11743 12271 12844 13306 Control *The acronym RFUrefers to units of relative fluorescence.

The increasing fluorescence seen in the experimental reaction correlatesto a decreasing opacity of the reaction mixture itself, which isperceivable with the naked eye. The decreasing opacity of the testreaction mixture was evident in the reaction vessels as a progressiveclearing of color from the reaction mixture, which was noticed as ofabout one minute of light exposure. In contrast, in the negativecontrol, the substantially constant relative fluorescence units seen inthe experiment correlate to the substantially constant opacity of thereaction mixture. Indeed, the negative control reaction mixture appearedto substantially maintain the same intensity of color through the entiretime course of the experiment.

The fluorescence noted in the experimental reaction emanates from thepainted bottom of the microtiter wells, which wells were identical tothose in which the negative controls were run. Accordingly, thecharacteristic that is actively changing in the reaction is theabsorbance, which, as it decreases in the test reaction mixture overtime, reveals more of the fluorescent paint coated onto the well. Afurther conclusion arising from this example is that one does not haveto measure an optical property that derives from the chemical state of adye; instead, one can more simply measure, or, truly, merely notice anuncovering of a second optical property of the reaction mixture and/orreaction vessel that contains the reaction mixture. The optical propertyof the dye included in the reaction mixture obscures a well-chosensecond optical property of the reaction mixture and/or reaction vessel.To the extent that the chemical state of the dye alters in the reactionmixture such that a decreasing concentration of the original dye remainsover time of the assay, the optical property contributed by the dye tothe reaction mixture diminishes, thereby revealing the presence of thesecond optical property, which can be used to trigger realization of thepresence or quantity of a target polynucleotide.

Example 17

This example sets forth an investigation of molecular weight changes ina dye over the course of a diagnostic reaction according to the presentinvention.

A mass spectroscopic analysis of a dye exposed to light in the presenceor absence of a ncPNA:DNA hybrid was accomplished, as follows:

Using a 96-well clear, streptavidin-coated microtiter plate (Nalge NuncInternational, Rochester, N.Y.; NUNC Immobilizer™ Streptavidin plates),a biotinylated ncPNA (i.e., biotin-(oo)-TGCCTCCCGTAG [SEQ ID NO:9]) wasused to capture isolated E. coli DNA. The PNA used in this experiment isspecific for a ubiquitous bacterial 16S sequence. To prepare themicroplate used in the experiment, the following steps were undertaken:(1) each well was washed three times with 300 μL phosphate-bufferedsaline solution with 0.05% Tween® 20 (“PBST”); (2) 50 μL aliquots of asolution containing 2.5 μL of 2 μM biotinylated ncPNA (5 pmoles), 5 μLof 10 ng/μL E. coli genomic DNA (50 ng), and 42.5 μL of 5 mM phosphatebuffer (pH 5.5) were introduced into wells of the microplate; (3) themicroplate was sealed and placed on a rotamixer for 60 minutes (at roomtemperature) to allow biotin-streptavidin interactions to occur; and (4)the wells were aspirated and washed five times with 200 μL PBST toremove unbound DNA and ncPNA.

A DiSC₂(3) solution was made by diluting an 8 mM (in DMSO) stocksolution to 2 mM with 5 mM phosphate buffer (pH 5.5). The dye solutionwas further diluted to 80 μM with a 5 mM phosphate/0.05% Tween® 20solution; aliquots of the diluted dye solution were introduced into eachwell. The plate was then placed on top of an Aurora 50/50 light for 30minutes before the solutions were pooled into a 15 mL conical containerwith an aluminum foil shroud, thereby keeping ambient light from thecontained solution. In order to have enough reaction product for liquidchromatography-mass spectrometry (“LC/MS”) experiments, 12 identicalwells were prepared for each reaction and pooled. Also included werecontrol wells with ncPNA only and with DNA only. Only those wells thatwere exposed to light and a ncPNA:DNA hybrid displayed the expectedreduction in fluorescence associated with the diagnostic method of thepresent invention.

The pooled products were then further analyzed by LC/MS using standardmethods and instrumentation via the services of a commercial analyticalchemistry laboratory. No ncPNA:DNA-specific dye product could be found,which was determined by comparison to LC analysis of products of the“Dye Only” control wells. However, an LC sample fraction collected at9.8 minutes included a new product of apparent molecular weight 427.1mass units (“mu”). The original parent dye compound, DiSC₂(3), can berepresented by C₂₁H₂₁N₂S₂, which is 365.5 mass units not including theiodide counterion. One new product of 427.1 mu corresponds to an oxygenmolecule (+15.56 mu) added to the parent dye compound, whichsubsequently forms an adduct with formic acid (+46.01 mu) during theliquid chromatography run. The new product has the empirical formula ofC₂₁H₂₁N₂OS₂.

Addition of the oxygen apparently disrupted conjugation within the dyemolecule rendering it colorless. The exact mechanism(s) of this reactionremains to be elucidated.

Example 18

This example is illustrative of a smartDNA reaction in a gel matrix,bound to a protein (or large macromolecule). Agarose super-shift assayswere performed. A biotinylated ncPNA (biotin-OO-TGCCTCCCGTAG [SEQ IDNO:9]) was hybridized to a complementary DNA oligonucleotide(5′-CTACGGGAGGCA-3′ [SEQ ID NO:57]) at a final concentration of 25 μM.Goat anti-biotin antibody (Immunology Consultants Laboratory, ICL) at 1mg/mL was used undiluted. In a microfuge tube, 1 μL of ncPNA:DNA duplexwas mixed with 1 μL antibody and allowed to sit at room temperature forthirty minutes to permit biotin-streptavidin interactions. Forcomparison, a second and third tube contained 1 μL ncPNA:DNA duplex onlyor antibody only, respectively. Each of these solutions was mixed with 5μL of a 50% glycerol solution and loaded into the wells of a 1% agarose(1×TBE) gel containing 2.5 μM DiSC₂(3) (added while the agarose wasmolten). Electrophoresis proceeded at 50V for 60 minutes in 1×TBErunning buffer. An initial time zero photograph was taken of the gelilluminated with a UV transilluminator. The gel was then exposed to alight stimulus Aurora 50/50 for 5 minutes before a second photograph wastaken.

Lanes 1 and 2 show “holes” (loss of fluorescence) for a fast migratingspecies corresponding to a ncPNA:DNA duplex (unbound to antibody)breaking down the dye after 5 minutes of exposure to light. Lane 1 showsa (super-shift) slower migrating “hole” suggesting that the ncPNA:DNAduplex is bound to the antibody through a biotin-streptavidininteraction and that this interaction does not interfere with thephotobleaching. Lane 3 (antibody only) shows no photobleaching therebyconfirming that the super-shift “hole” in Lane 1 is not due to theantibody.

Future experiments include chemically coupling the ncPNA:DNA duplex toan antibody which is specific for a given antigen.

Example 19

This example illustrates an embodiment of the present invention withvarying lengths of ncPNA.

Six different 17-mer and 12-mer ncPNA probes targeting similar nucleicacid sequences within isolated Mycobacteria tuberculosis (MTB) genomicDNA were tested.

Working solutions of 2 μM ‘12-mer nccocktail’ were generated from equalvolumes of 2 μM solutions of each individual 12-mer ncPNA. Workingsolutions of 2 μM ‘17-mer nccocktail’ were generated from equal volumesof 2 μM solutions of each individual 17-mer ncPNA. The PNAs used are setforth in Table 14 below.

TABLE 14 SEQ ID Code Name PNA Sequence NO: TB01biotin-(OO)-GTCGTCAGACCCAAAAC 36 TB02 biotin-(OO)-CGAGAGGGGACGGAAAC 37TB03 biotin-(OO)-TGAACCGCCCCGGCATG 38 TB04 biotin-(OO)-ACCAAGTAGACGGGCGA39 TB05 biotin-(OO)-CATCCAACCGTCGGTCG 40 TB06biotin-(OO)-ACAAGACATGCATCCCG 41 TB07 lysine-CAGACCCAAAAC 42 TB08lysine-CGAGAGGGGACG 43 TB09 lysine-TGAACCGCCCCG 44 TB10lysine-ACCAAGTAGACG 45 TB11 lysine-CATCCAACCGTC 46 TB12lysine-ACAAGACATGCA 47

Introduced into a 384-well white with white bottom microtiter plate(purchased from Costar) were: 2 μL of a 1 ng/μL solution of MTB genomicDNA (obtained from MRL, Department of Microbiology, Ft. Collins, Colo.),2 μL of the 2 μM ‘cocktail’ of 12-mer ncPNAs or 2 μL of the 2 μM‘cocktail’ of 17-mer PNAs, and 16 μL H₂O (Molecular Biology Grade). Thissolution was vigorously mixed for 5 seconds and allowed to incubate for10 minutes at room temperature. Thirty microliters of a solutioncontaining 5 mM phosphate (pH 5.5), 0.083% Tween® 80, and 15 μMdiethylthiacarbocyanine iodide (DiSC₂(3)) was then added to each well(final concentration of 3 mM phosphate, 0.05% Tween® 80, 9 μM DiSC₂(3))and mixed.

An initial T₀ reading (excitation 535 nm, emission 590 nm) was obtainedusing a Tecan Genios fluorescence microtiter plate reader. Next, thereactive mixtures in the microtiter wells were exposed to light using anAurora 50/50 light. Fluorescence readings were taken every two minutesout to 30 minutes total light exposure time. To assess non-specificbinding of ncPNAs, an identical experiment containing human genomic DNA(isolated from a B cell line (GM 14686; Coriell Cell Repositories,Camden, N.J.)) instead of MTB DNA was run in parallel. Control wellscontaining ncPNA only (no DNA) and DNA only (no ncPNA) were alsoincluded. The average fluorescence of four identical reactions wasplotted along with standard error bars as a function of light exposuretime.

The data indicate that ncPNAs having 12 or 17 nucleotides are usefullyemployed with the present invention. Whereas the rate of change influorescence was indeed faster with the larger ncPNA targeting MTB DNA(see FIG. 17A), a greater level of non-specific activity was shown indata generated using the larger PNA (see FIG. 17B).

The data are consistent with the view that a ‘cocktail’ of 12-mer ncPNAscan drive a diagnostic reaction after incubation with isolated MTBgenomic DNA at room temperature. Although a ‘cocktail’ of 12-mer ncPNAcorrelates to a slower rate than a ‘cocktail’ of 17-mer PNAs, fewernon-specific reactions were detected when the 12-mer PNAs were combinedwith the unrelated DNA as compared to when the 17-mer PNAs were combinedwith the unrelated DNA.

Example 20

This example illustrates the use of individual ncPNAs, as opposed to a“cocktail” of multiple different ncPNAs in concert, to detect MTB DNA.

In particular, ncPNA TB11 [SEQ ID NO:45] and ncPNA TB12 [SEQ ID NO:47]were used in the protocol set forth in Example 14 to detect isolatedgenomic MTB DNA at 2, 1, 0.5, 0.25, 0.0625, and 0 ng/50 μL reactions(with the exception that the “DNA Standards” were diluted in 10 mMTris-Cl (pH 7.2), 1 mM EDTA, 0.05% Tween-80 buffer).

Briefly, 25 μL of the “DNA Standards” (0.08 ng/μL, 0.04 ng/μL, 0.02ng/μL, 0.01 ng/μL, 0.005 ng/μL, 0.0025 ng/μL, and 0 ng/μL) weredispensed into a 384-well white with clear bottom microtiter plate (NUNCcatalog #242763). From an 8.3 μM stock (prepared in H₂O), ncPNA TB10 (orncPNA TB 12) was diluted to a working concentration of 53.3 nM inTris-Cl (pH 7.2), 1 mM EDTA, 0.05% Tween® 80 buffer. This solution wasmixed with equal volumes of a solution containing 36 μM DiSC₂(3) inTris-Cl (pH 7.2), 1 mM EDTA, 0.05% Tween® 80 buffer. Twenty-fivemicroliters of this mixture were added to the “DNA standard” in themicrotiter, followed by centrifugation of the plate, shaking, andincubation at room temperature as per the protocol in Example 14.

Absorbance measurements at 556 nm were recorded at T₀ and every twominutes thereafter, up to 44 minutes of photoactivation. Photoactivationwas done using a solid-state activator providing illumination at 470 nmwith a power density of 2 mW/cm². Data was compiled and assessed for thetime at which absorbance had reached 50% of initial starting absorbance(defined as the T₅₀%) as depicted in the Table 15 below.

TABLE 15 Time to reach 50% absorbance, (T_(50%)) in minutes amount ofDNA per well ncPNA TB10 ncPNA TB12 2 ng 11.3 10 1 ng 14.6 12.5 0.5 ng16.7 16.5 0.25 ng 23.2 23.7 0.125 ng 28.3 21.7 0.0625 ng 32.1 28.2 ncPNAOnly (No DNA) 34.2 33.1 2 ng DNA Only 38.6 33.9 (No ncPNA)

The reactions containing ncPNA TB12 had lower T₅₀% times as compared toncPNA TB 10, suggesting faster detection of MTB DNA. This difference inperformance between the two ncPNAs could be attributed to eithersequence-specificity difference (of the particular ncPNA:DNA duplexesformed), or a targeting ability (strand-invasion into a genomic region),or a target copy number difference of each ncPNA. While ncPNA TB 10 hasthe potential to bind upwards of 20 sequences within the MTB genome(IS6110 transposon sequence of variable copy number), ncPNA TB 12 onlyhas a single genomic target (rDNA), but is also specific for MTB rRNAthat may be present (residual) in the genomic DNA preparations.

Example 21

The following example illustrates the present invention in oneembodiment where DNA is used as a “detector” for assays involvingphotoactivation of light-sensitive dyes.

In photoactivation reactions, interaction of the dye with a chargedpolymer is important for increasing light-sensitivity of the dye. Thisexperiment demonstrates that the amount of DNA present in a reaction (inthe absence of a nucleic acid analog) can accelerate photoactivation ofthe dye. This suggests that photoactivation of the dye can be used todetect quantities of unknown DNA in a solution, if compared to astandard curve of known quantities of DNA. It also suggests that DNAalone (without hybridizing a nucleic acid analog) can be used as adetector if coupled to a ligand that participates in receptor-ligandinteractions in solid-support formats.

Lyophilized calf thymus DNA (Sigma Catalog #D4764) was dissolved in 10mM Tris, 1 mM EDTA (pH 7.5) to concentrations of 19.5 ng/μL. From thissolution, a series of two-fold dilutions were created down to aconcentration of 0.075 ng/μL.

Into wells of a 384-well white microtiter plate (Greiner) were dispended2 μL of each DNA dilution along with 20 μL of molecular grade H₂O. Anadditional 30 μL of a 5 mM phosphate buffer (pH 5.5) with NP-40 (0.05%)with DiSC2(3) (final 9 μM) was introduced into the well. An initialfluorescence measurement (excitation at 535 nm, emission at 590 nm) wastaken with a Tecan Genios microtiter plate reader. The wells werephotoactivated with the Aurora 50/50 and fluorescent readings wererecorded every two minutes. Table 16 lists the relative fluorescenceunits for each DNA concentration every two minutes.

TABLE 16 time exposed to RFUs of photoactivation assays of increasingamounts of DNA light (min) 39 ng 19.5 ng 9.8 ng 4.9 ng 2.4 ng 1.2 ng 0.6ng 0.3 ng 0.15 ng 0 9236 10912 11763 12066 12331 12441 12580 12506 126812 4076 9424 11391 11786 12005 12138 12294 12252 12325 4 3538 6119 1014111152 11419 11669 11803 11795 11817 6 3792 4344 8541 10633 11093 1143111602 11602 11649

As can be seen from the data in Table 15, the higher DNA concentrations(39 ng/well and 19.5 ng/well) gave an initial (T₀) of lower RFUsrelative to the lower DNA concentrations. In addition, the higher DNAconcentrations also caused a greater rate of decrease in RFUs during thefirst 6 minutes of photoactivation. This “DNA standard curve” provides acomparison against solutions containing unknown amounts of DNA.

Alternatively, when the DNA is linked to a protein or ligand, the DNAacts as a “detector” for protein-receptor or ligand-receptorinteractions on solid support formats where no extraneous DNA isexpected to be present. DNA remaining on the solid support (followingwashes) (through indirect binding) cause photobleaching of the dye uponexposure to a photoactivator.

Example 22

This example illustrates one embodiment of the present invention forcomparison of different cyanine dyes and using LED at differentwavelengths in smartDNA reaction.

2 types of probe-nucleic acid (NA) target systems were used in thestudy. The first one was referred as oligo system, which includes a18mer DNA and its complementary PNA probe. The 18mer DNA has thesequence: 5′-ACGCACAATCCCACTATC-3′ (SEQ ID NO: 20), and was ordered fromIDTDNA. The DNA was resuspended in molecular biology grade water(Hyclone, catalog #SH30538.03) for freezer storage, and initiallyquantified by converting the absorbance of the NA solution at 260 nm(measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. Stock solution of the 18merin H₂O at 250 nM was prepared and stored in 4° C. and ready to use.

PNA bio18, with the sequence of: GATAGTGGGATTGTGCGT from N to C terminiand with biotin tag at the N-terminus (SEQ ID NO: 1) was used as theprobe for the oligo system. A 50 μM PNA probe mix was prepared fromfreezer stocks, and from this stock a 250 nM PNA stock in H₂O wasfurther prepared and stored at 4° C. and ready for use.

Another screening system was referred as the genomic system in thepresent example. Samples containing or not containing genomic nucleicacid (NA) isolated from Mycobacterium tuberculosis (MTB CDC1551) wereprepared and tested using a protocol of the present invention.Accordingly, the samples were used in generating reaction mixtures thatfurther included a dye and a nucleic acid analog that specificallyhybridizes to MTB DNA. Optical properties of the reaction mixture beforeand after exposure to a light source were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.08 ng/μL in TE buffer(Tris-C110 mM, pH 7.2, 1 mM EDTA, with 0.05% Tween-80).

For the genomic system, a PNA mixture of 12mer PNAs were used as theprobe. A 50 μM PNA probe mix was prepared from freezer stocks. Sequencesused were SEQ ID NOS: 42, 43, 44, 45, 46 and 47. Freezer stocks (at 200μM in H₂O) were put in a hotblock at 65° C. for five minutes, and a 50μM solution of each ncPNA was prepared by diluting the freezer stockncPNA in molecular biology grade H₂O (Hyclone, catalog #SH30538.03).Equal volumes of each 50 μM ncPNA solution were added to create a 50 μMtotal PNA concentration, i.e., 8.33 μM per ncPNA. This 50 μM PNA probemix was put in the hotblock at 65° C. for five minutes, and was thenfurther diluted in TE buffer (Tris-C110 mM, pH 7.2, 1 mM EDTA) withsurfactant (0.05% Tween-80) to a final concentration of 320 nM. The 320nM PNA probe mix was placed in a water bath at 65° C. for five minutes,removed, and given 30 minutes to cool to room temperature.

Various dye stock solutions were prepared, including the standard dye insmartDNA assay, 3,3′-diethylthiacarbocyanine(“DiSC₂(3)”). For thestandard dye, the stock was prepared by diluting 7.5 mM DiSC₂(3)(solubilized in DMSO) into phosphate buffer with surfactant (0.05%Tween-80) to a working concentration of 36 μM. Other dyes tested includederivatives of the standard dye. Among them a few examples were3,3′-di(propyl, allyl, butyl or pentyl) thiacarbocyanine (thesubstituents variations), 3,3′-diethyloxycarbocyanine, and3,3′-diethylthia(di, tri)carbocyanine.

For the oligo system, 4 reaction mixtures in triplicate were set up foreach dye, for each wavelength of LED exposure. The first well containsonly the dye in buffer, and was referred as “dye only.” The second wellcontains dye and the PNA probe (biol8) and was referred as “PNA only.”The third well contains dye and the NA target (either the oligo or thegenome), and was referred as “DNA only.” The forth well contains dye,the NA target and the corresponding PNA probe and was referred as“PNA/DNA.” The final concentration in the reaction mixture for dye was 9μM, PNA 100 nM, DNA 100 nM, or PNA/DNA 50 nM, if applicable.

For genomic system, Serial 1:2 dilutions of the 0.08 ng/μL MTB NA (in 10mM TE buffer, with 0.05% Tween-80) were prepared down to a lowconcentration of 0.0025 ng/μL; referred to herein as “DNA standards.”Aliquots of 25 μL of each of the DNA standards were dispensed into a 384well white/clear plate (NUNC, catalog #242763) to set up a concentrationcurve with triplicate in individual columns on the plate. The columnswere set up as follows: columns 1-6, 9-14 and 17-22 were assay wellswith 2 ng, 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, and 0.0625 ng MTB DNA perwell, column 7, 15 and 23 was a control well with 0 ng DNA and column 8,16 and 24 was a control well with 2 ng MTB DNA.

After the buffer or the DNA standards were dispensed into themicroplate, and the microplate was centrifuged, two dye mixtures werethen prepared. The reaction set-up steps were performed in a dimly litroom. The first control dye solution was prepared by mixing equalvolumes of 36 μM DiSC₂(3) with TE buffer (with 0.05% Tween-80.) This 18μM DiSC₂(3) control solution was mixed directly in a reagent reservoir.A 25 μL aliquot of the control dye solution was then dispensed to “dyeonly” and “DNA only” wells on the microplate. The second, dye+PNA probemix was prepared by mixing equal volumes of 36 μM DiSC₂(3) with the 320nM 12mer PNA probe mix (for the genomic system). This 18 μM DiSC₂(3)+160nM PNA probe was mixed directly in a reagent reservoir. A 25 μL aliquotof the dye+PNA probe mix was then dispensed to “PNA only” and “PNA/DNA”wells on the microplate giving final concentrations of 9 μM and 80 nMfor the dye and PNA, respectively. The microplate was then brieflycentrifuged at 500 RPM.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. For the oligo system, after the initial optical measurement, theplate was removed from the microplate reader, and subjected tophotoactivation for 10 minutes. The light for photoactivation wasprovided by a solid-state activator providing illumination withdifferent peak wavelength of: 390 nm (425 μW/cm²) (LumexSSL-LX5093SUVC), 410 nm (446 μW/cm²) (Lumex SSL-LX5093UVC), 470 nm (1400μW/cm²) (Jameco #183222), 490 nm (1340 μW/cm²) (Marubeni AmericaCorporation, L490-33U), 505 nm (1300 μW/cm²) (Jameco #334473), 515 nm(1000 μW/cm²) (Jameco #183214), 574 nm (124 μW/cm²) (Toshiba TLGE158P),620 nm (423 μW/cm²) (Liteon Electronics Inc, 2F3VAKTN), 640 nm (2060μW/cm²) (Liteon Electronics Inc, 2F3VRKTN), and 660 nm (948 μW/cm²) (QTOptoelectronics MV8114), as measured with a laser-based power meterhaving the tradename LaserCheck™ (Coherent, Inc., Santa Clara, Calif.;catalog #0217-271-00). The microplate was reinserted into the reader andeach well was measured as indicated above, however with a shortenedorbital shake and settle time of one second. Then the plate was removedfrom the microplate reader and subjected to photoactivation foradditional 50 minutes. The microplate was reinserted into the reader andeach well was measured as indicated above.

For the genomic system, after the initial optical measurement the platewas removed from the microplate reader and subjected to photoactivationfor a two minute interval. The microplate was reinserted into the readerand each well was measured as indicated above, however with a shortenedorbital shake and settle time of one second each. This two minutephotoactivation followed by absorbance detection cycle was performedover a period of 60 minutes total exposure to light.

Dyes that were photobleached after 60 minutes preferentially in PNA/DNAsamples were selected as potential candidates for smartDNA reaction.Table 17 summarizes the optical properties of the dyes when exposed todifferent wavelength of LEDs (at different intensities) in the 4reaction mixtures. For those dyes that were photobleached after 60minutes preferentially in PNA/DNA samples, the wavelength of thespecific working LED is given.

TABLE 17 Dye number, chemical name, λ_(max) for absorbance and observedoptical properties when exposed to LED in the 4 reaction mixture usingoligo system.. In the description of response to LED exposure, “nodifference” means there was no difference in absorbance change of atleast one controls, “dye only”, “PNA only”, or “DNA only” comparing with“PNA/DNA” samples. Dye Maximum Response to LED Number Chemical NameAbsorbance(nm) Exposure 1 3,3′-Diethylthiacarbocyanine 554 470, 490,505, and iodide 515 nm, works fast, 5-10 min. 2 3,3′-Diethyl-9- 540non-specific decrease, methylthiacarbocyanine iodide 390-515 nm allphotobleaching. 3 3,3′-Diethylthiacyanine iodide 422 no difference,robust to various LEDs. 4 3,3′-Diethylthiadicarbocyanine 648 nodifference, but iodide photobleached by 505-660 53,3′-Diethylthiatricarbocyanine 754 no difference, but iodidephotobleached by 505-660 6 3,3′-Diethylthiatricarbocyanine 752 nodifference, but perchlorate photobleached by 505-660 73,3′-Diallylthiacarbocyanine 556 470, 505, and 515 nm Bromide 83,3′-Diethyloxacarbocyanine 480 no difference, robust to iodide variousLEDs. 9 3,3′-Diethyl-2,2′- 386 no difference, robust toOxathiacarbocyanine Iodide various LEDs. 10 3,3′-Dimethyloxacarbocyanine480 no difference, robust to iodide various LEDs. 113,3′-Diethyloxadicarbocyanine 578 no difference, iodide photobleached by505, 515. 12 3,3′-Dipropylthiacarbocyanine 556 470, 505, and 515 nmiodide 13 3,3′-Dipropylthiadicarbocyanine 650 no difference, but iodidephotobleached by 620, 640 and 660 nm 14 3,3′-Dipropyloxacarbocyanine 482no difference, robust to iodide various LEDs. 153,3′-Dibutylthiacarbocyanine 556 470 and 490 nm. iodide 163,3′-Dipentylthiacarbocyanine 556 470 and 490 nm. iodide 173,3′-Dihexyloxacarbocyanine 554 no difference, robust to iodide variousLEDs. 18 1,1′-Diethyl-2,2′-cyanine iodide 522 no difference, robust tovarious LEDs. 19 1-1′-Diethyl-2,2′-carbocyanine 600 505 and 515 nmiodide 20 1,1′-Diethyl-2,2′-carbocyanine 600 505 and 515 nm bromide 211,1′-Diethyl-4,4′-carbocyanine 698 no difference, but iodidephotobleached by 640 and 660 nm 22 1,1′-Diethyl-3,3,3′,3′- 544 nodifference, robust to tetramethylindocarbocyanine various LEDs. iodide23 1,1′-Dipropyl-3,3,3′,3′- 544 no difference, robust totetramethylindocarbocyanine various LEDs. iodide 24 1-[[3-Ethyl-2(3H)-548 no difference, robust to benzothiazolylidene]ethylidene]- variousLEDs. 2(1H)-naphthalenone 25 3,3′-Diethylthiacyanine 422 no differenceethylsulfate 26 3-Ethyl-9-methyl-3′-(3- 540 no difference, butsulfatobutyl) thiacarbocyanine photobleached by betaine 470 nmLED. 273-Carboxymethyl-3′,9-diethyl- 548 no difference, but5,5′-dimethylthiacarbocyanine photobleached by betaine 470 nmLED. 283,3′-Diethyloxatricarbocyanine 680 no difference, decay iodide 29thiazole orange 502 no difference, but photobleached by 470 nmLED. 301,1′-Diethyl-2,4′-cyanine iodide 556 no difference 31[5-[2-(3-Ethyl-3H-benzothiazol-2- 540 no differenceylidene)-ethylidene]-4-oxo-2- thioxo-thiazolidin-3-yl]-acetic acid 321-Butyl-2-[3-(1-butyl-1H- 680-750 no differencebenzo[cd]indol-2-ylidene)- propenyl]-benzo[cd]indolium tetrafluoroborate33 1,3,3-Trimethyl-2-(2-[2- 782 no difference, decayphenylsulfanyl-3-[2-(1,3,3- trimethyl-1,3-dihydro-indol-2-ylidene)-ethylidene]-cyclohex-1- enyl]-vinyl)-3H-indolium chloride 344,5,4′,5′-Dibenzo-3,3′-diethyl-9- 644 no difference, decaymethyl-thiacarbocyanine bromide

For the standard dye DiSC₂(3), the absorbance change in the reactionmixtures when exposed to the different LEDs, and in oligo system for“DNA only” and “PNA/DNA” reaction mixtures were measured and summarizedin Tables 18, 19, and 20. “PNA only” and “dye only” mixtures always hada similar or lower changes in absorbance compared with “DNA only”controls and thus were not listed here.

TABLE 18 Absorbance change of reaction mixture for “DNA only” (DNA +wavelength) and “PNA/DNA” (PD + wavelength) after exposure of LEDwavelengths: 390 nm, 410 nm or 490 nm. DNA390 PD390 DNA410 PD410 DNA490PD490 0 0.51 0.51 0.51 0.51 0.52 0.52 10 0.48 0.43 0.49 0.48 0.31 0.1060 0.39 0.21 0.38 0.28 0.17 0.05

TABLE 19 Absorbance change of reaction mixture for “DNA only” (DNA +wavelength) and “PNA/DNA” (PD + wavelength) after exposure of LEDwavelengths: 470 nm, 505 nm or 515 nm. DNA470 PD470 DNA505 PD505 DNA515PD515 0 0.52 0.52 0.52 0.52 0.52 0.52 10 0.32 0.13 0.31 0.12 0.33 0.1960 0.15 0.04 0.11 0.04 0.13 0.04

TABLE 20 Absorbance change reaction mixture for “DNA only” (DNA +wavelength) and “PNA/DNA” (PD + wavelength) after exposure of LEDwavelength: 620 nm, 640 nm or 660 nm. DNA620 PD620 DNA640 PD640 DNA660PD660 0 0.43 0.43 0.43 0.43 0.52 0.44 10 0.40 0.38 0.39 0.34 0.50 0.4260 0.32 0.24 0.27 0.07 0.44 0.33

From the above data, it was demonstrated that 470 nm and 505 nm LED werethe 2 wavelengths that resulted the higher rate in photobleaching for“PNA/DNA” samples. Another wavelength worth notice was the 640 nm LED,which also resulted selectively photobleaching of “PNA/DNA” samples.

Standard curve experiments were performed on DiSC₂(3) with 470 nm or 640nm LEDs as the light source. The results of the slope of dye colorchange at different NA amount per 50 μl reaction mixture for 470 nm and640 nm LED were summarized in Table 21.

TABLE 21 Slope of dye color change (mAbsorbance/min for the first 4min.) with 0, 0.0625 ng, 0.125 ng, 0.25 ng, 0.5 ng, 1 ng and 2 ng MTBgenomic NA in homogeneous smartDNA reactions, exposed to 470 nm or 640nm LED. 470 nm 640 nm 0 11.8 1.6 0.0625 ng 13.9 1.9 0.125 ng 13.2 2.50.25 ng 18.1 2.8 0.5 ng 29.0 3.7 1 ng 47.9 6.1 2 ng 64.1 7.7

From Table 21, it was demonstrated that 470 nm was the more efficientLED exposure source for smartDNA assay, as the reaction rate for dyecolor change was much higher.

Example 23

This example illustrates one embodiment of the present invention forcomparison of LEDs with different intensity in smartDNA assay.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) were prepared and testedusing a protocol of the present invention. Accordingly, the samples wereused in generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.08 ng/μL in phosphatebuffer (10 mM) with surfactant (0.05% Tween-80). All reagents wereprepared using molecular biology grade water (Hyclone, catalog#SH30538.03).

At this point, a 50 μM PNA probe mix was prepared from freezer stocks.Sequences used were SEQ ID NOS: 42, 43, 44, 45, 46 and 47. Freezerstocks (at ˜200 μM in H₂O) were put in a hotblock at 65° C. for fiveminutes, and a 50 μM solution of each ncPNA was prepared by diluting thefreezer stock ncPNA in molecular biology grade H₂O (Hyclone, catalog#SH30538.03). Equal volumes of each 50 μM ncPNA solution were added tocreate a 50 μM total PNA concentration, i.e., 8.33 μM per ncPNA. This 50μM PNA probe mix was put in the hotblock at 65° C. for five minutes, andwas then further diluted in phosphate buffer (10 mM) with surfactant toa final concentration of 320 nM. The 320 nM PNA probe mix was placed ina water bath at 65° C. for five minutes, removed, and given 30 minutesto cool to room temperature.

Four dye stock solution were prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) intophosphate buffer with surfactant to working concentrations of 18, 36, 60and 80 μM, and were referred as 4× dye stocks.

Serial 1:2 dilutions of the 0.08 ng/μL MTB NA (in 10 mM TE buffer, with0.05% Tween-80) were prepared down to a low concentration of 0.0025ng/μL; referred to herein as “DNA standards”. Aliquots of 25 μL of eachof the DNA standards were dispensed into a 384 well white/clear plate(NUNC, catalog #242763) to set up a concentration curve with triplicatein individual columns on the plate. The columns were set up as follows:columns 1-6, 9-14 and 17-22 were assay wells with 2 ng, 1 ng, 0.5 ng,0.25 ng, 0.125 ng, and 0.0625 ng MTB DNA per well, column 7, 15 and 23was a control well with 0 ng DNA and column 8, 16 and 24 was a controlwell with 2 ng MTB DNA. 4 rows of such were prepared.

For each of the 4 dye stock solutions, two dye mixtures were thenprepared. The reaction set-up steps involving the dye were performed ina dimly lit room. The first control dye solution was prepared by mixingequal volumes of each of the 4× DiSC₂(3) with phosphate buffer (with0.05% Tween-80.) The resulted 2× DiSC₂(3) control solution were mixeddirectly in a reagent reservoir. A 25 μL aliquot of the control dyesolution was then dispensed to each well along column 8 and othertriplicate wells of the microplate, respectively. The second, dye+PNAprobe mix was prepared by mixing equal volumes of each of the 4×DiSC₂(3) with the 320 nM PNA probe mix. This 2× DiSC₂(3)+160 nM PNAprobe mix was mixed directly in a reagent reservoir. A 25 μL aliquot ofthe dye+PNA probe mix was then dispensed to each well along column 1-7and all the other triplicates of the microplate, in respective rows.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm, withdifferent power densities provided by power supply voltages of 12V,13.8V, 15V and 18V yielding 4 different power densities on themicroplate. The power supply voltages yielded power densities of 1.8,1.1, 0.7, and 0.32, 0.7, 1.1, and 1.8 mW/cm², respectively, as measuredwith a laser-based power meter having the tradename LaserCheck™(Coherent, Inc., Santa Clara, Calif.; catalog #0217-271-00). Themicroplate was reinserted into the reader and each well was measured asindicated above, however with a shortened orbital shake and settle timeof one second each. This two minute photoactivation followed byabsorbance detection cycle was performed over a period of 10 to 20minutes total exposure to light.

Results of standard smartDNA assay, in homogenous solutions, with 4different dye concentrations, and at 4 different power outputs weresummarized in Tables 22, 23, 24, and 25 below.

TABLE 22 Slope of dye color change (mAbsorbance/min) for 4.5 μM dye atvarious LED power supply. DNA amount(ng) 18 V 15 V 13.8 V 12 V 0 8.3 77.7 1.9 0.0625 8 5.6 5.1 1.9 0.125 8.7 6.7 7.1 1.9 0.25 11.4 9 8.8 3.30.5 16.4 13.7 12.8 5.3 1 24.9 21.5 17.3 8.6 2 35.3 30.4 24.3 12.5

TABLE 23 Slope of dye color change (mAbsorbance/min) for 9 μM dye atvarious LED power supply. DNA amount(ng) 18 V 15 V 13.8 V 12 V 0 18 13.913.7 2.3 0.0625 17.7 13.4 12.4 4.1 0.125 19.3 15 14 4.1 0.25 23 18.318.4 5.6 0.5 31.4 25.5 24.6 9.6 1 45.3 38.5 34.3 14.9 2 60.1 51.4 45.421

TABLE 24 Slope of dye color change (mAbsorbance/min) for 15 μm dye atvarious LED power supply. DNA amount(ng) 18 V 15 V 13.8 V 12 V 0 33.923.9 22.4 5.5 0.0625 31.9 23.4 20.4 7.1 0.125 34.3 25 22.2 7.1 0.25 37.627.3 24.8 8.5 0.5 46 34.8 31.2 12.1 1 61.1 48.4 41.9 16.1 2 75.4 61.452.3 21.9

TABLE 25 Slope of dye color change (mAbsorbance/min) for 20 μM dye atvarious LED power supply. DNA amount(ng) 18 V 15 V 13.8 V 12 V 0 45.1 3430.5 7.2 0.0625 43.3 34 28.7 8.8 0.125 46.2 34 32.2 9.7 0.25 49 34.9 3610.8 0.5 55.8 41.1 39.7 13.5 1 68.8 53.6 49.6 17.6 2 87.2 65.7 55.5 23.2

The results demonstrated higher bleaching rate at higher LED power forthe same NA amount and same dye concentration, but also increased thebackground bleaching rate. Same trend was observed for higher dyeconcentration, where the background bleaching rate was also increased.At the experimental conditions, a dye concentration between 9 μM to 15μM was the optimal concentration range, where the background bleachingwas not increased significantly, and a power supply from 15V to 18V wasthe optimal LED power supply range.

Example 24

This example illustrates one embodiment of the present invention foreffects of different detergents in smartDNA assay.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) (Colorado StateUniversity) were prepared and tested using a protocol of the presentinvention. Accordingly, the samples were used in generating reactionmixtures that further included a dye and a nucleic acid analog thatspecifically hybridizes to MTB DNA. Optical properties of the reactionmixture before and after exposure to a light source were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.04 ng/μL in 1 mM EDTA.All reagents were prepared using molecular biology grade water (Hyclone,catalog #SH30538.03).

A 50 μM solution of each ncPNA was prepared by diluting the freezerstock ncPNA in molecular biology grade H₂O. The sequence of the PNA wasTGA ACC GCC CCG GCA TG from N to C terminus, with 1 biotin at the Nterminus (TB03; SEQ ID NO: 38). This 50 μM PNA probe stock was furtherdiluted in homopipes buffer (pH 5.0, 20 mM) without surfactant to afinal concentration of 320 nM. The 320 nM PNA probes were stored in 4°C. and used at room temperature.

Detergents used in this study were purchased from Pierce, catalogue #28328, which contains 10% stock solutions or solid power of thefollowing detergents: Tween-80, Tween-20, Triton 110, Triton 114, Brij35, Brij 58, NP40, Chaps, octyl glucoside, octylthio glucoside, andcetyltrimethylammonium bromide (CTAB). The detergents were used directlyfrom the 10% stock, or 10% stock was prepared by dissolving the solidpower in molecular grade H₂O.

Dye stock was prepared by diluting 7.5 mM 3,3′-diethylthiacarbocyanine(“DiSC₂(3)”) (solubilized in DMSO) into homopipes buffer withoutsurfactant to a working concentration of 36 μM.

For each of the detergents, a series of dilutions in the 0.04 ng/μL MTBNA (in 1 mM EDTA) were prepared, so that the final concentration of thedetergents in the reaction mixture would be 0, 0.02%, 0.04%, 0.08%,0.12%, 0.16% and 0.2%. Aliquots of 25 t of the solutions were dispensedinto a 384 well white/clear plate (NUNC, catalog #242763) to set up aconcentration curve for each of the detergent with triplicate inindividual columns on the plate. Thus, the final mixture would containthe expected amount of a certain detergent, and 1 ng of MTB NA. (1 ng ofMTB NA corresponds to approximately 200,000 genomes). Duplicate rowswere set up for each detergent as described above.

After the DNA dilutions with the increasing amount of detergents weredispensed into the microplate, 2 dye mixtures were then prepared. Thereaction set-up steps involving the dye were performed in a dimly litroom. The first control dye solution was prepared by mixing equalvolumes of 36 μM DiSC₂(3) with the 10 mM homopipes buffer (pH 5.0, nosurfactant). This 18 μM DiSC₂(3) control solution was prepared directlyin a reagent reservoir. Dye+PNA probe mix was prepared by mixing equalvolumes of 36 μM DiSC₂(3) with 320 nM PNA probe in 10 mM homopipesbuffer. A 25 μL aliquot of the control solution (dye only) was dispensedto all wells of the first row of the duplicate rows, and dye+PNA probemix was then dispensed to each well of the second row. This gave finalconcentrations of 9 μM and 80 nM dye and ncPNA, respectively.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 20 minutes totalexposure to light.

Table 26 shows the change in absorbance based on the first 4 minutesupon exposure to light (milliAbsorbance/minute) for each detergent ateach concentration (average of triplicate reactions). Each detergent has2 rows of data. The first row data corresponds to the DNA+PNA+dyesolution, and the second row corresponds to the dye+PNA solution.

TABLE 26 Slope of dye color change (in mAbs/min) for increasing amountsof detergent in a homogenous smartDNA assay using TB03, Homopipes pH 5.0buffer with or without DNA. Detergent 0.00% 0.02% 0.04% 0.08% 0.12%0.16% 0.20% Tween-80 34.7 22.8 20.2 15.2 11.8 9.1 7.5 Tween-80 37.6 27.323.3 19.9 14.7 12.0 10.1 Tween-20 40.9 40.9 27.3 23.5 18.0 14.6 12.1Tween-20 41.8 41.8 29.6 26.0 22.3 18.4 13.7 Triton-100 41.0 27.8 21.913.4 9.7 7.9 6.8 Triton-100 39.2 29.0 24.3 14.8 9.5 8.0 6.7 Triton-11442.9 34.1 30.8 25.7 19.9 16.1 11.8 Triton-114 42.4 34.7 33.1 27.3 21.819.0 14.0 Brij-35 41.2 26.6 22.7 15.5 10.6 8.0 6.8 Brij-35 41.4 31.226.9 19.5 14.1 11.0 8.0 Brij-58 43.8 24.2 20.3 10.8 8.6 6.3 5.4 Brij-5844.0 27.2 24.2 16.4 12.5 9.3 6.6 NP40 43.5 25.3 20.5 12.7 9.9 8.0 6.5NP40 45.2 26.9 22.2 14.5 10.4 7.4 6.2 CHAPS 42.6 37.5 33.5 32.0 30.229.3 28.8 CHAPS 45.5 38.3 36.1 35.5 34.5 33.5 31.6 Octyl glucoside 33.641.2 39.3 36.4 34.7 31.7 31.2 Octyl glucoside 37.4 47.5 46.3 43.8 40.939.2 39.0 Octylthio glucoside 34.1 0.0 39.5 41.3 40.9 37.9 38.0Octylthio glucoside 38.5 40.0 44.1 44.3 46.8 44.7 45.6 SDS 37.5 16.818.6 24.7 29.9 26.5 19.1 SDS 42.0 39.9 20.4 24.4 27.8 21.4 19.7 CTAB38.4 24.8 23.9 22.9 21.3 19.8 17.4 CTAB 39.9 27.6 25.3 24.4 20.9 19.118.2

Optimal detergents for smartDNA reaction demonstrate the greatestdifferential in change in absorbance between reactions with and withoutDNA, without significantly decreasing the overall rate of the smartDNAreaction. Tween-80 is the standard detergent for smartDNA assay becauseat a concentration between 0.04% to 0.08% there was an increaseddifference between the control row (no PNA) and the sample row (withPNA). The same trend was also true for Brij 58 (at about 0.08%) andTween-20 (at about 0.08%) because at these conditions they contributedto decreased background photobleaching rate of the dye, while minimizinginterference with the specific smartDNA photobleaching reaction.

Example 25

This example illustrates one embodiment of the present invention fordetermining the optimal buffer conditions and optimal PNA sequences inthe smartDNA detection. In this example the homogeneous assay is used.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) were prepared and testedusing a protocol of the present invention. Accordingly, the samples wereused in generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed. S. pneumoniae (ATCC# BAA-3340) and Human DNA (fromSigma, lot #123K₃₇₉₆) were used as nonspecific NA.

The concentration of isolated MTB NA (in water for freezer storage) andnonspecific DNA was initially quantified by converting the absorbance ofthe NA solution at 260 nm (measured with a Hewlett-Packard Model NO:HP8452A diode-array spectrophotometer) using standard methods. For thereaction mixtures, the nonspecific and specific NA were diluted down toa concentration of 0.04 ng/μL in 1 mM EDTA. All reagents were preparedusing molecular biology grade water (Hyclone, catalog #SH30538.03).

Series of buffers with various pH were prepared. Phosphate-citratebuffer was prepared by mixing stock solutions of 0.2M Na₂HPO₄ (fromTeknova, catalogue # S0215) and 0.1M citric acid (from Teknova,catalogue # C2440) to a final pH of 4.0, 4.2, 4.4, 4.6 and 4.8.Homopipes buffers were prepared by titrating 20 mM homopipes using 4%NaOH to a final pH of 4.0, 4.2, 4.4, 4.6, 4.8 and 5.0. Tris-EDTA buffer1M stock of pH 7.2, 7.4, 8.0, 8.5 and 9.0 were purchased from Teknova(catalogue # T5072, T5074, T5080, T5085, T5090, respectively) anddiluted to working concentration with molecular biology grade H₂O forusage.

At this point, a 50 μM PNA probe stock was prepared from freezer stocks.Sequences used were TB01: GTC GTC AGA CCC AAA AC (SEQ ID NO: 36), TB03:TGA ACC GCC CCG GCA TG (SEQ ID NO: 38), TB04: ACC AAG TAG ACG GGC GA(SEQ ID NO: 39), and these 3 have a biotin tag at the N-terminus; TB24:GTC GTC AGA CCC AAA AC, with 1 lysine at the N and another lysine at theC terminus (SEQ ID NO: 57). All sequences are from N to C terminus. The50 μM PNA probes were further diluted in buffers with different pH asdescribed above with surfactant (0.1% Tween-80) to a final concentrationof 320 nM, 640 nM and 1280 nM. The 320 nM, 640 nM and 1280 nM probeswere stored in 4° C. and used at room temperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) intophosphate buffer with surfactant to a working concentration of 36 μM.

Aliquots of 25 μL of 0.04 ng/μL MTB NA, 25 μL of the 0.04 ng/μLnonspecific Human DNA, and 25 μL of 1 mM EDTA (as control) weredispensed into a 384 well white/clear plate (NUNC, catalog #242763) toset up a set of triplicate in individual columns on the plate. Thecolumns were set up as follows: 1 ng MTB DNA, 1 ng Human DNA, 1 ng S.pneumoniae and EDTA blank.

After the DNA standards were dispensed into the microplate, for each PNAsequence, in each kind of buffer, five dye mixtures were then prepared.The reaction set-up steps involving the dye were performed in a dimlylit room. The first control dye solution was prepared by mixing equalvolumes of 36 μM DiSC₂(3) with a specific buffer (with surfactant (0.1%Tween-80)) as described. This 18 μM DiSC₂(3) control solution was mixeddirectly in a reagent reservoir. A 25 μL aliquot of the control dyesolution was then dispensed to each well along the specific NA,nonspecific NA and control wells on the microplate. The other, dye+PNAprobe mix was prepared by mixing equal volumes of 36 μM DiSC₂(3) withthe 320, 640 and 1280 nM PNA probe mix. This 18 μM DiSC₂(3)+160, 320 and640 nM PNA probe mix prepared directly in a reagent reservoir. A 25 μLaliquot of each of the dye+PNA probe mix was then dispensed to thespecific NA, nonspecific NA and control wells on the microplate.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 20 minutes totalexposure to light.

The results of the reaction rate in the first 4 minutes in the units ofmilliAbsorbance per minute for each of the specific (MTB) andnonspecific NA (human) with TB01 (SEQ ID NO: 36), and at increasing PNAconcentrations were summarized in Table 27. The buffers werephosphate-citrate buffer series.

TABLE 27 Slope of dye color change (mAbsorbance/min) with 1 ng MTB NAand 1 ng human NA in homogeneous smartDNA reactions with different TB01(SEQ ID NO: 36) PNA concentrations and buffer pH. PNA ConcentrationpH4.0 pH4.4 pH4.8 MTB 13.3 nM   19.9 28.0 12.0 40 nM 18.8 41.7 15.6 80nM 12.9 49.4 28.0 160 nM  14.8 64.8 18.4 Human 13.3 nM   15.3 23.9 14.240 nM 16.1 26.4 17.0 80 nM 16.9 31.2 26.6 160 nM  19.3 43.1 20.1

The results of the reaction rate in the first 4 minutes in the units ofmilliAbsorbance per minute for each of the specific (MTB) andnonspecific NA (human) with TB03 (SEQ ID NO: 38), and at increasing PNAconcentrations were summarized in Table 28. The buffers used werehomopipes buffer series.

TABLE 28 Slope of dye color change (mAbsorbance/min) with 1 ng MTB NAand 1 ng human NA in homogeneous smartDNA reactions with different TB03(SEQ ID NO: 38) PNA concentrations and buffer pH. pH4.0 pH4.2 pH4.4pH4.6 pH4.8 pH5.0 MTB  0 14.7 12.1 11.1 15.6 13.1 14.9  80 nM 22.8 2322.7 38.9 38.1 40.6 160 nM 21.0 24.8 26.9 43.3 46.4 53.2 320 nM 15.427.3 29.1 41.6 51.5 56.9 Hu-  0 18.4 15.1 14.5 18.0 16.3 18.0 man  80 nM21.2 18.4 18.7 28.7 24.2 22.6 160 nM 18.5 19.5 21.3 29.2 23.8 25.2 320nM 16.4 20.6 24.8 26.9 26.3 26.8

The results of the reaction rate in the first 4 minutes in the units ofmilliAbsorbance per minute for each of the specific (MTB 1 ng/reaction)and nonspecific NA (human 1 ng/reaction) with TB04 (SEQ ID NO: 39), andat increasing PNA concentrations were summarized in Table 29, with thespecific MTB NA and the Human nonspecific NA. The buffers used werehomopipes buffer series.

TABLE 29 Slope of dye color change (mAbsorbance/min) with 1 ng MTB NAand 1 ng human NA in homogeneous smartDNA reactions with different TB04(SEQ ID NO: 39) PNA concentrations and buffer pH. pH4.0 pH4.2 pH4.4pH4.6 pH4.8 pH5.0 MTB  0 13.7 12.4 13.7 15.6 11.9 13.2  80 nM 29.9 33.833.1 39.4 35.8 36.4 160 nM 32.7 38.7 41.5 50.8 47.4 49.5 320 nM 34.441.3 51.8 57.5 57.1 61.2 Hu-  0 16.4 14.8 16.3 16.5 13.8 14.9 man  80 nM15.8 16.5 15.2 21.3 17.9 18.1 160 nM 16.2 17.2 19.1 22.5 18.2 20.2 320nM 17.8 18.3 23.9 22.2 21.8 25.8

The results showed that for the above 3 PNA probes with biotin tags havean optimal pH. Increasing the concentration of PNA probes also increasethe reaction rates. Both non-specific NA did not show interference insmartDNA assay.

The results of the reaction rate in the first 4 minutes in the units ofmilliAbsorbance per minute for each of the specific and nonspecific NAwith TB24 (SEQ ID NO: 57), and at increasing PNA concentrations weresummarized in Table 30, with the specific MTB NA 1 ng/reaction and thenonspecific S. pneumoniae NA 1 ng/reaction. The buffers used wereTris-EDTA buffer series.

TABLE 30 Slope of dye color change (mAbsorbance/min) with 1 ng MTB NAand 1 ng S. pneumoniae in homogeneous smartDNA reactions with differentTB24 (SEQ ID NO: 57) PNA concentrations and buffer pH. pH7.2 pH7.4 pH8pH8.5 pH9 MTB 0 19.1 15.8 18.1 14.7 15.3  80 nM 65.8 74.0 72.2 78.0 84.9160 nM 82.1 83.4 114.9 87.4 83.8 S. pneumoniae  0 14.0 14.0 16.7 14.512.9  80 nM 17.1 22.0 16.2 17.2 20.3 160 nM 22.6 21.4 32.9 18.2 21.2

The results showed that for TB24 (SEQ ID NO: 57), the PNA with 1 lysineat the N and 1 lysine at the C terminus, pH impacts the reactionsensitivity and specificity and the optima is above pH 7. Increasing theconcentration of PNA probes also increase the reaction rates.Non-specific S. pneumoniae NA did not show interference in smartDNAassay.

Example 26

This example illustrates one embodiment of the present invention forsensitivity comparison of PNA probes that have the same sequence butinclude different number and position of lysine residues.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) were prepared and testedusing a protocol of the present invention. Accordingly, the samples wereused in generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.04 ng/μL in 1 mM EDTA(diluted from 500 mM EDTA, Teknova catalogue # E0306). All reagents wereprepared using molecular biology grade water (Hyclone, catalog#SH30538.03).

At this point, a 50 μM PNA probe was prepared from freezer stocks. Thesequence of the PNA was GTC GTC AGA CCC AAA AC from N to C terminus,with 1 lysine at the N terminus (TB 14; SEQ ID NO: 58); 2 lysines: 1 atthe N terminus and the other at the C terminus (TB24; SEQ ID NO: 57); or4 lysines: 2 at the N terminus and the other 2 at the C terminus (TB44;SEQ ID NO: 59). A 50 μM solution of each PNA was prepared by dilutingthe freezer stock PNA in molecular biology grade H₂O, using 1.5 mLprotein Lo-bind tubes (Eppendorf, catalogue # 0030108116). This 50 μMPNA probe was further diluted in homopipes buffer (pH 5.0, 20 mM,prepared from Homopipes solid powder, Research Organics # 6047H) or TEbuffer (pH 7.2, 20 mM Tris-Cl (diluted from 1M stock, Teknova catalogue# T5072), 2 mM EDTA) without surfactant to a final concentration of 640nM. The 640 nM PNA probes were stored in 4° C. and used at roomtemperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”)(from SigmaAldrich, catalogue #173738) (solubilized in DMSO, Sigma # D8418) into homopipes buffer or TEbuffer with surfactant (0.2% Tween-80, from 10% stock, Pierce Biotech, #28328) to a working concentration of 36 μM.

Serial dilutions using the 0.04 ng/μL MTB NA (in 1 mM EDTA) wereprepared, and aliquots of 25 μL of each of the DNA dilutions weredispensed into a 384-well white with optical bottom microplate (NUNC,catalog #242763) to set up a concentration curve with triplicate inindividual columns on the plate. The final amount of MTB NA in each wellcorresponds to approximately 80000, 40000, 20000, 10000, 5000 and 0genomes per reaction (1 ng of MTB NA corresponds to approximately 200000genomes).

After the DNA dilutions were dispensed into the microplate, dye mixtureswere then prepared. The reaction set-up steps involving the dye wereperformed in a dimly lit room. Dye+PNA probe mix was prepared by mixingequal volumes of 36 μM DiSC₂(3) in homopipes buffer or TE buffer witheach of the 640 nM PNA probe in corresponding buffer. All 18 μMDiSC₂(3)+320 nM PNA probe mixtures were prepared directly in a reagentreservoir (Diversified Biotech, RESE 2000). A 25 μL aliquot of thedye+PNA probe mix was then dispensed to each well containing the DNAdilutions.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 20 minutes totalexposure to light.

The results of the reaction rate in the first 4 minutes in the units ofmilliAbsorbance per minute for each of the NA dilutions with each of thevarious PNA probes were summarized in Table 31 and Table 32.

TABLE 31 Slope of dye color change for increasing amount of MTB genomesin a homogenous smartDNA assay using TB14 (SEQ ID NO: 58), HomopipespH5.0 buffer or TB24 (SEQ ID NO: 57), TE pH7.2 buffer as probe. STDEVdata for the TB24 (SEQ ID NO: 57) as probe were also listed. App.genomes TB14 (SEQ ID TB24 (SEQ ID STDEV/TB24 (SEQ per reaction NO: 58)NO: 57) ID NO: 57) 0 16.7 18.4 0.21 5000 17.9 20.6 0.63 10000 18.9 21.90.23 15000 20.4 24.1 1.85 20000 20.4 26.0 0.76 30000 22.2 31.3 2.3240000 26.0 35.8 3.13 80000 37.0 51.3 1.66

TABLE 32 Slope of dye color change for increasing amount of MTB genomesin a homogenous smartDNA assay using TB14 (SEQ ID NO: 58) (in homopipespH 5.0), TB24 (SEQ ID NO: 57) (in TE pH 7.2 buffer) or TB44 (SEQ ID NO:59) (in TE pH 7.2 buffer) as probe. App. genomes per TB14 (SEQ ID TB24(SEQ ID TB44 (SEQ ID reaction NO: 58), HP5.0 NO: 57), TE7.2 NO: 59),TE7.2 0 15.3 16.7 21.6 25000 25.4 35.8 43.2 50000 30.8 45.0 47.8 10000043.2 62.0 68.2 200000 51.9 93.1 100.8

The results show that by attaching 1 lysine to the N terminus and 1lysine to the C terminus of the PNA probe the sensitivity of thedetection (in TE buffer) was increased (the slope of the reaction rate(mAbs/min) vs. genomes per reaction was 42 compared with 25 changes ofunits per 100000 genomes). However, attaching 4 lysines, 2 each at the Nand C terminus of the PNA probe did not change the sensitivity of theassay compared with the PNA with 1 lysine at each end in the same TEbuffer.

Example 27

This example examines the relationship between PNA-DNA complexconcentrations and the rate of turnover of the dye during the photobleaching reaction. This analysis suggests that the PNA-DNA complex actsin a catalytic manner. Preparation and activation of reaction mixturescontaining M. tuberculosis DNA (MTB DNA), PNA, and dye have beenpreviously described. Absorbance vs. time data for these analyses weretaken from FIG. 18. Turnover rates for the dye were calculated using thefollowing procedure and illustrated in Table 33. For each concentrationof MTB DNA and the PNA only control the initial rate of the reaction wascalculated by subtracting the absorbance of the reaction solutionobtained at 4 minutes from the initial value and dividing by 4. Initialabsorbances of reaction solutions were normalized to a common minimumvalue using the following relationship,NA(t)=A(t)−(A(t ₀)−A(mint ₀))

Where NA(t) is the normalized value of the absorbance at any time t.A(t) is the uncorrected absorbance at time t. A(t₀) is the initialabsorbance of each sample, and A(min t₀) is the initial absorbance valueof the sample having the lowest absorbance. All other startingabsorbances were normalized to this value. This procedure eliminatesslight differences in the starting absorbances of the reaction solutionsand allows a more straightforward comparison of turn over rates derivedfrom these values. Initial rates of absorbance change were thenconverted to the number of molecules of dye turned over per minute bydividing the absorbance change by the molar absorptivity (1.36 E+05 cm⁻¹M⁻¹) of the dye and then multiplying the result by the reaction volume(5.0E−05 1) and Avogadro's number (6.023 E+23). Since the dye photobleaches in the presence of PNA alone this background rate of bleachingwas then subtracted from the rates for each of the MTB DNAconcentrations. The approximate number of MTB genomes was calculatedfrom each sample amount using a genome size for MTB of 4411532 bp. ThePNA probes used in this experiment can theoretically bind to 89 sites inthe MTB genome, the number of potential catalytic sites was derived bydividing total genomes present by 89. This represents the lowestturnover number for the catalytic sites present. If not all sites arefully occupied with PNAs or functional the actual turnover number willbe higher. Turnover numbers calculated in this manner are remarkablyuniform in value and appear to have a constant value over the 30 foldvariation in analyte concentration averaging 2.0 E+03 dyemolecules/second with 16% relative standard deviation. This isconsistent with the notion that the PNA-DNA complex can function as acatalyst. The turnover numbers obtained are also consistent with thoseobtained for common enzymes which are shown in Table 34.

TABLE 33 Dye Turnover Per Catalytic Site as a Function of AnalyteConcentration MTB Amount (ng) 2    1.0000 0.5000 0.2500 0.1250 0.0625PNA Initial Absorbance (normalized) 0.4859 0.4859 0.4859 0.4859 0.48590.4859 0.4859 Absorbance at 4 minutes 0.3771 0.4195 0.4460 0.4589 0.46310.4646 0.4671 Delta Absorbance (0-4 min) 0.1088 0.0664 0.0399 0.02700.0228 0.0213 0.0188 Divide by dye molar absorptivity = 1.36E+05 8.0E−074.9E−07 2.9E−07 2.0E−07 1.7E−07 1.6E−07 1.4E−07 (molarity) Divide by 4(molar/min) 2.0E−07 1.2E−07 7.3E−08 5.0E−08 4.2E−08 3.9E−08 3.5E−08Multiply by reaction volume 50 ul (moles/min) 1.0E−11 6.1E−12 3.7E−122.5E−12 2.1E−12 2.0E−12 1.7E−12 Multiply by 6 × 10²³ (molecules/min)6.0E+12 3.7E+12 2.2E+12 1.5E+12 1.3E+12 1.2E+12 1.0E+12 Subtract out PNAbackground (molecules/min) 5.0E+12 2.6E+12 1.2E+12 4.6E+11 2.2E+111.4E+11 0.0E+00 TB Genomes (CDC1551 4403837bp) 4.1E+05 2.1E+05 1.0E+055.2E+04 2.6E+04 1.3E+04 — PNA sites (for 6 PNA cocktail) 8.9E+01 8.9E+018.9E+01 8.9E+01 8.9E+01 8.9E+01 — Catalytic Sites 3.7E+07 1.8E+079.2E+06 4.6E+06 2.3E+06 1.2E+06 — Dye Turnover/Site (molecules/min)1.4E+05 1.4E+05 1.3E+05 9.9E+04 9.6E+04 1.2E+05 — Dye Turnover/Site(molecules/sec) 2.3E+03 2.4E+03 2.1E+03 1.6E+03 1.6E+03 2.0E+03 —

TABLE 34 Turnover Rate of Common Enzymes Turnover Number Enzyme(molecules/second) Carbonic Anhydrase 6.0E+05 Acetylcholinesterase2.5E+04 Amylase 1.8E+04 Penicillinase 2.0E+03 DNA Polymerase 1.5E+01

Example 28

This example illustrates one embodiment of the present invention foreffects of different preservatives in smartDNA assay.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) were prepared and testedusing a protocol of the present invention. Accordingly, the samples wereused in generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. For the reaction mixtures,the NA was diluted down to a concentration of 0.08 ng/μL in phosphatebuffer (10 mM) with surfactant (0.05% Tween-80). All reagents wereprepared using molecular biology grade water (Hyclone, catalog#SH30538.03).

At this point, a 50 μM PNA probe mix was prepared from freezer stocks.Sequences used were SEQ ID NOS: 42, 43, 44, 45, 46 and 47. Freezerstocks (at 200 μM in H₂O) were put in a hotblock at 65° C. for fiveminutes, and a 50 μM solution of each ncPNA was prepared by diluting thefreezer stock ncPNA in molecular biology grade H₂O (Hyclone, catalog#SH30538.03). Equal volumes of each 50 μM ncPNA solution were added tocreate a 50 μM total PNA concentration, i.e., 8.33 μM per ncPNA. This 50μM PNA probe mix was put in the hotblock at 65° C. for five minutes, andwas then further diluted in phosphate buffer (10 mM) with surfactant(0.05% Tween-80) to a final concentration of 320 nM. The 320 nM PNAprobe mix was placed in a water bath at 65° C. for five minutes,removed, and given 30 minutes to cool to room temperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) intophosphate buffer with surfactant (0.05% Tween-80) to a workingconcentration of 36 μM.

Serial 1:2 dilutions of the 0.08 ng/μL MTB NA (in 10 mM TE buffer, with0.05% Tween-80) were prepared down to a low concentration of 0.0025ng/μL; referred to herein as “DNA standards”. Aliquots of 25 μL of eachof the DNA standards were dispensed into a 384 well white/clear plate(NUNC, catalog #242763) to set up a concentration curve with triplicatein individual columns on the plate. The columns were set up as follows:columns 1-6, 9-14 and 17-22 were assay wells with 2 ng, 1 ng, 0.5 ng,0.25 ng, 0.125 ng, and 0.0625 ng MTB DNA per well, columns 7, 15 and 23were control wells with 0 ng DNA and columns 8, 16 and 24 were controlwells with 2 ng MTB DNA. 3 rows of such were prepared.

For the first row, two dye mixtures were then prepared. The reactionset-up steps involving the dye were performed in a dimly lit room. Thefirst control dye solution was prepared by mixing equal volumes of 36 μMDiSC₂(3) with phosphate buffer (with 0.05% Tween-80.) This 18 μMDiSC₂(3) control solution was mixed directly in a reagent reservoir. A25 μL aliquot of the control dye solution was then dispensed to eachwell along column 8 and all other triplicate of the microplate. Thesecond, dye+PNA probe mix was prepared by mixing equal volumes of 36 μMDiSC₂(3) with the 320 nM PNA probe mix. This 18 μM DiSC₂(3)+160 nM PNAprobe mix was mixed directly in a reagent reservoir. A 25 μL aliquot ofthe dye+PNA probe mix was then dispensed to each well along columns 1-7and all the other triplicates of the microplate.

For the second and third row, similar dye mixtures were then prepared.For both the 18 μM DiSC₂(3) control solution and the dye+PNA probe mix,either sodium azide(NaN₃) was added to a final of 0.2% from 2% stock(100 μl 2% NaN₃ stock per 1 ml of mixture), or Proclin 300 was added toa final of 0.1% from 3.49% stock solution (from SUPELCO, catalogue #48119-U, and 28.6 μl 3.49% Proclin 300 stock per 1 ml of mixture). 25 μLaliquot of the final dye solution was then dispensed to each well alongeach column as described above.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 26 minutes totalexposure to light.

The results for adding preservatives into the standard smartDNA assaywas summarized in Table 35.

TABLE 35 Slope of dye color change (mAbsorbance/min) for increasingamount of MTB NA, without the presence of any preservatives, or in thepresence of 0.1% NaN₃, or in the presence of 0.05% Proclin300. DNAamount(ng) control NaN3 Proclin300 0 11.7 10.7 12.3 0.0625 12.6 11.412.7 0.125 14.2 11.9 14.5 0.25 17.9 13.1 17.4 0.5 23.5 14 23.7 1 36.315.8 34.7 2 48 19.1 46.2

Table 35 showed that the presence of 0.1% NaN₃ interfered withhomogenous smartDNA assay, as the rate of dye color change is similar asbackground bleaching rate, and much lower than the control assay.However, the presence of 0.05% Proclin300 retains the same slope of dyecolor change in all DNA amounts. Thus 0.05% Proclin300 can be used as apreservative in the storage of smartDNA buffers.

Example 29

The microparticle-captured PNA:oligo assay was performed using PNA Bio18(N-biotin-OOOOO-GATAGTGGGATTGTGCGT-C(PNA); SEQ ID NO: 1) and itscomplementary oligo (5′-ACGCACAATCCCACTATC-3′; SEQ ID NO: 20).

In a 1.5 mL microcentrifuge tube, 10 picomoles binding capacitystreptavidin polystyrene particles (Spherotech, catalog #SVP-40-5) werewashed with 1 mL capture buffer (10 mM Tris pH 7.2, 1 mM EDTA, 150 mMNaCl, 0.05% Tween® 80); followed by centrifugation at 14,000 rpm for 2minutes to pellet the microparticles. Washing was repeated 2 times.

Fifty μM PNA of SEQ ID NO: 1 and DNA of SEQ ID NO: 20 or bio-DNA(5′-biotin-TTTTT-GATAGTGGGATTGTGCGT-3′; SEQ ID NO: 60) solutions weremade by diluting concentrated stock solutions with molecular biologygrade water (Hyclone, catalog #SH30538.03). As a control, a biotinylatedDNA oligo with the same sequence as the PNA (SEQ ID NO: 60) was alsoincluded. Five μL of PNA solution (or biotinylated DNA oligo), 5 μLcomplementary DNA solution and 40 μL of hybridization buffer (10 mM TrispH 7.2, 1 mM EDTA, 20 mM NaCl) were mixed and heated 95° C. in a heatblock. The block was removed from the heater and allowed to cool to roomtemperature on the benchtop. Four μL PNA:DNA hybridization mixture (20pmoles) or 4 μL biotinylated-DNA:DNA hybridization mixture was added to96 μL of capture solution containing 10 pmole binding capacity of beads.This was set this up so that there were 10 reactions worth of PNA:DNAduplex and beads in the same tube. The tubes were gently agitated for1.5 hours at room temperature to capture the biotinylated-PNA:DNA hybridand the biotinylated-DNA:DNA to the streptavidin microparticles. Themicroparticles were then washed once with capture buffer as describedabove; followed by two washes with 1×HP4.8 buffer (10 mM Homopipes(Research Organics catalog #6047H) pH 4.8, 0.05% Tween® 80 (PierceBiotechnology, catalog #28328). The microparticles were then resuspendedin final volume of 500 μL 1×HP4.8 buffer, 0.5 mM EDTA and 9 μM DiSC₂(3).

The following reaction set-up steps involving the dye were performed ina dimly lit room. Aliquots of 50 μL of each of the samples weredispensed in triplicate sets into a 384-white-well optical bottommicrotiter plate (NUNC, catalog #242763). The microplate was insertedinto the Tecan Safire² monochromator-based microplate reader. Themicroplate was subjected to a 1 second medium-intensity orbital shake,followed by a 1 second settle time, followed by an absorbancemeasurement at 556 nm. The absorbance measurement was performed with abandwidth of 20 nm, with 12 reads per well. After the initial opticalmeasurement, the plate was removed from the microplate reader, andsubjected to photoactivation for a two minute interval. The light forphotoactivation was provided by a solid-state activator providingillumination with a peak wavelength of 470 nm and a power density ofapproximately 1.8 mW/cm², as measured with a laser-based power meterhaving the tradename LaserCheck™ (Coherent, Inc., Santa Clara, Calif.;catalog #0217-271-00). The microplate was reinserted into the reader andeach well was measured as indicated above, however with a shortenedorbital shake and settle time of one second each. This two minutephotoactivation followed by absorbance detection cycle was performedover a period of 20 minutes total exposure to light.

The initial rates of dye photobleaching (initial dye color change rates)were calculated by subtracting the absorbance of the solution attime_(4min) from the initial absorbance of the solution at Time_(0min)and dividing by 4 minutes. Table 36 lists the initial dye color changerates depicted as change in milliabsorbance (mAbs) units per minute.

TABLE 36 Standard Deviation of Initial Change in Dye Initial Dye ColorSample Absorbance Change Absorbance PNA:DNA hybrid 60.28 9.11 PNA alone24.82 2.23 DNA alone 24.61 2.77 Bio-DNA:DNA hybrid 40.22 2.28 Bio-DNAalone 39.38 3.13 DNA alone 25.27 1.44 microparticles only 34.65 1.38

Example 30

This example illustrates one embodiment of the present invention fordetermining the LOD of isolated MTB genomic nucleic acid (NA) in thecapture and release assay.

Samples containing or not containing target NA isolated fromMycobacterium tuberculosis (MTB CDC1551) were prepared and tested usinga protocol of the present invention. These samples were then used ingenerating reaction mixtures that further included a dye and a nucleicacid analog that specifically hybridizes to MTB DNA. Optical propertiesof the reaction mixture before and after exposure to a light source wereobserved.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. A stock of 8 ng/μl NAsolution was prepared from original stock using molecular biology gradewater (Hyclone, catalog #SH30538.03).

At this point, a 50 μM PNA probe mix was prepared from freezer stocks.Sequence used was GTC GTC AGA CCC AAA AC from N to C terminus, with abiotin at the N-terminus (SEQ ID NO: 36) (for the capture portion of theassay), or 1 lysine at the N terminus and 1 lysine at the C terminus(SEQ ID NO:57) (for detection in the homogeneous released portion of theassay). A 50 μM solution of each ncPNA was prepared by diluting thefreezer stock ncPNA in molecular biology grade H₂O (Hyclone, catalog#SH30538.03). This 50 μM PNA probe was further diluted in TE buffer (pH7.2, 20 mM Tris-Cl, 2 mM EDTA) without surfactant to a finalconcentration of 640 nM. The 640 nM PNA probe stock was stored in 4° C.and used at room temperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) into 20mM TE buffer with surfactant to a stock concentration of 36 μM.

PNA microparticles were prepared by conjugating biotin tagged PNA tostreptavidin polystyrene particles (Spherotech, catalog #SVP-40-5), inmicroparticle coating buffer (150 mM NaCl, 1× Tris-EDTA, pH7.2, 0.05%Tween-80). Aliquot of 200 pmoles capture capacity of particles waswashed 3× in coating buffer, resuspended in 500 ul coating buffer, and 8ul of 50 uM of biotin tagged PNA was added. The mixture was shaking atroom temperature for 3 hrs. After coating with PNA, the microparticleswas washed 3×, first with coating buffer as described above, then twicewith 2× homopipes (pH5.0) buffer, and stored in 1 mM EDTA at the finalconcentration of 20 pmole/50 ul for use.

20 pmoles (4×) of the PNA microparticles were used in each sample, andwere divided to triplicate reaction mixtures on the micro titer plate.Thus, 5 pmoles binding capacity of the microparticles was used in eachreaction mixture. Equal volume of dye stock was mixtured with themicroparticles in EDTA. From a NA stock of 0.1 ng/μl (approximately20000 genomes per μl), MTB NA were added to the microparticles and dyemixture, with final MTB amount 0, 20000, 40000, 80000, 160000 and 320000genomes per tube. These amounts were four times the number of genomes inthe final reaction tubes, thus the eluate was used in four reactionswith 0, and approximately 5000, 10000, 20000, 40000 and 80000 genomesper reaction mixture in each well on the micro-titer plate

The mixture was then incubated and shaken at room temperature in thedark for 30 minutes. After incubation, the microparticles were washed 3×with washing buffer (0.5 mM EDTA, 1× homopipes buffer, 0.05% Tween 80, 9μM dye).

100 of 1 mM EDTA was added to the washed microparticles, pipetted toresuspend the pelleted microparticles, and the mixture incubated on thebenchtop for 30 minutes to elute the captured NA on the microparticles.

The 100 ul mixture was then spun for 2 minutes to pellet themicroparticles. The supernatant was aliquoted directly to microtiterplate, with 25 ul for each reaction mixture in triplicate. After theeluates were dispensed into the microplate, dye mixtures were thenprepared. The following reaction set-up steps involving the dye wereperformed in a dimly lit room. Dye+PNA probe mix was prepared by mixingequal volumes of 36 μM DiSC₂(3) with the 320 nM PNA probe mix. This 18μM DiSC₂(3)+160 nM PNA probe mix was mixed by inversion in a 15 mLconical tube, and poured into a reagent reservoir. A 25 μL aliquot ofthe dye+PNA probe mix was then dispensed to each well containing theelution.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 44 minutes totalexposure to light.

The results of the reaction rate in the first 4 minutes in the units ofmille Absorbance per minute for each of the NA capture samples with thePNA probes were summarized in Table 37.

TABLE 37 Slope of dye color change for different amount of targetgenomes in homogenous smartDNA assay. App. genomes per reaction reactionrate(mAbs/min) STDEV 0 17.0 0.22 5000 18.4 0.24 10000 17.3 0.26 2000022.6 0.66 40000 26.6 0.27 80000 35.3 0.71

These results suggested that isolated MTB can be captured to the PNAmicroparticles and released for smartDNA detection. Using 5 times STDEVabove background to estimate the LOD, LOD of isolated MTB NA in thecaptured assay is 5000 genomes in the 50 ul reaction mixture.

Example 31

This example illustrates one embodiment of the present invention fordetecting specific isolated DNA in the presence of non-specific DNA fromvarious microorganisms. In this example the capture and release formatis employed.

Samples containing or not containing genomic nucleic acid (NA) isolatedfrom Mycobacterium tuberculosis (MTB CDC1551) were prepared and testedusing a protocol of the present invention. Accordingly, the samples wereused in generating reaction mixtures that further included a dye and anucleic acid analog that specifically hybridizes to MTB DNA. Opticalproperties of the reaction mixture before and after exposure to a lightsource were observed.

The concentration of isolated MTB NA (in water for freezer storage) wasinitially quantified by converting the absorbance of the NA solution at260 nm (measured with a Hewlett-Packard Model NO: HP8452A diode-arrayspectrophotometer) using standard methods. A stock of 32 ng/μl NAsolution was prepared from original stock. The concentration ofnon-specific NA from various microorganisms were also determined usingthe same methods. Nonspecific NA from microorganisms used in this studywere purchased from ATCC, and included: S. aureus (ATCC# 108320), S.pneumoniae (ATCC# BAA-3340), H. influenzae (ATCC# 51907D), E. coli(ATCC# 7009280), N. meningitidtis (ATCC# 108320), M. gordonae (ATCC#108320), P. aeruginosa (ATCC# 108320), K. pneumonae (ATCC# 108320).Nonspecific NA also included isolated Human DNA (from Sigma,lot#123K3796).

At this point, a 50 μM PNA probe mix was prepared from freezer stocks.Sequence used was GTC GTC AGA CCC AAA AC from N to C terminus, with alysine (SEQ ID NO: 58) or a biotin (SEQ ID NO: 36) at the N terminus. A50 μM solution of each ncPNA was prepared by diluting the freezer stockncPNA in molecular biology grade H₂O (Hyclone, catalog #SH30538.03).This 50 μM PNA probe was further diluted in homopipes buffer (pH 5.0, 20mM, referred as 2× buffer) without surfactant to a final concentrationof 320 nM. The 320 nM PNA probe mix was stored in 4° C. and used at roomtemperature.

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) into 20mM homopipes buffer with surfactant to a working concentration of 36 μM.

PNA microparticles were prepared by conjugating biotin tagged PNA tostreptavidin polystyrene particles (Spherotech, catalog#SVP-40-5), inmicroparticle coating buffer (150 mM NaCl, 1× Tris-EDTA, pH7.2, 0.05%Tween-80). Aliquot of 200 pmoles capture capacity of particles waswashed 3× in coating buffer, resuspend in 500 ul coating buffer, and 8ul of 50 uM of biotin tagged PNA was added. The mixture was shaking atroom temperature for 3 hours. After coating with PNA, the microparticleswas washed 3×, first with coating buffer, then twice with 2× homopipesbuffer, and stored in 1 mM EDTA at the final concentration of 20pmole/50 ul for use.

20 pmoles (4×) of the PNA microparticles were used in each sample, andwill be divided to triplicate reaction mixtures on the micro titerplate. Thus 5 pmoles binding capacity of microparticles will be used ineach reaction mixture. Equal volume of dye stock was mixed with themicroparticles in EDTA, 32 ng of specific and/or nonspecific NA wasadded to the microparticle and dye mixture, the mixture was thenincubated and shaken at room temperature in the dark for 30 minutes.After incubation, the microparticles was washed 3× with washing buffer(0.5 mM EDTA, 1× homopipes buffer, 0.05% Tween 80, 9 uM dye).

100 μl of 1 mM EDTA was added to the washed microparticles, pipetted toresuspend the pelleted microparticles, and the mixture was left on thebenchtop for 30 minutes to elute the captured NA off the microparticles.

The 100 ul mixture was then spun for 2 minutes to pellet themicroparticles, and the NA containing supernatant was aliquoted directlyto microtiter plate, with 25 μl for each reaction mixture in triplicate.After the eluates were dispensed into the microplate, dye mixtures wereprepared. The following reaction set-up steps involving the dye wereperformed in a dimly lit room. Dye+PNA probe mix was prepared by mixingequal volumes of 36 μM DiSC₂(3) with the 320 nM PNA probe mix. This 18μM DiSC₂(3)+160 nM PNA probe mix was mixed by inversion in a 15 mLconical tube, and poured into a reagent reservoir. A 25 μL aliquot ofthe dye+PNA probe mix was then dispensed to each well containing theelution.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 10 secondmedium-intensity orbital shake, followed by a 600 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 10 to 20 minutes totalexposure to light.

The results of the reaction rate in the first 4 minutes in the units ofmille Absorbance per minute for captured specific NA in the backgroundof nonspecific NA were summarized in Table 38.

TABLE 38 Slope of dye color change of the capture smartDNA assay, whenthe NA available for capture is nonspecific NA as listed below, or thenonspecific NA plus the specific MTB NA. Sample Without specific MTB NAWith Specific MTB NA Control (MTB) 20.0 129.0 Human DNA 17.7 125.2 S.aureus 24.8 126.7 S. pneumoniae 21.8 128.7 H. influenzae 19.0 121.7 E.coli 20.8 125.1 N. meningitotis 22.7 123.4 M. gordonae 33.6 123.7 P.aeruginosa 19.8 123 K. pneumonae 21.8 118.8

The results showed that isolated MTB DNA can be selectively captured tothe PNA microparticles in the background of equal amount of non-specificNA described above, and released for smartDNA detection. Theinterference NA summarized in Table 38 did not increase the backgroundof the capture assay (except M. gordonae). The capture of target MTB NAhad the same apparent efficiency whether interfering NA was present ornot.

Example 32

The presence of DNA captured using the smartDNA assay was confirmedusing polymerase chain reaction (PCR).

32 ng of genomic DNA from each Mycobacterium bovis (Genbank accessionnumber NC_(—)002945) and Mycobacterium tuberculosis was captured using20 picomoles binding capacity TB01 (SEQ ID NO: 36)-microparticles usingthe standard smartDNA capture assay. DNA was eluted using 10 μL ofmolecular biology grade water (Hyclone, catalog #SH30538.03). The DNAsolution was placed on ice and mixed with 4 μL 25 mM MgCl₂ (Promegacatalog #A3511), 10 μL 5× GoTaq Flexi Green buffer (Promega catalog#M8911), 0.25 μL GoTaq Flexi DNA polymerase (Promega, catalog #M8295), 2μL 10 mM dNTP mix (Promega, catalog #C1141), 2 μL 10 μM forward primer[5′-TGAACCGCCCCGGCATGTCC-3′ (SEQ ID NO: 61) for M. bovis and5′-CCGGTTAGGTGCTGGTGGTC-3′ (SEQ ID NO: 62) for M. tuberculosis] and 2 μL10 μM reverse primer [SEQ ID NO: 61] for M. bovis, and5′-CGCCCCATCGACCTACTACG-3′ (SEQ ID NO: 63) for M. tuberculosis] and20.75 μL of molecular biology grade water in a total reaction volume of50 μL. The cycling conditions were as follows: (1) 94° C. for 4 minutes;(2) 94° C. for 60 seconds; (3) 55° C. for 60 seconds; (4) 72° C. for 3minutes; (5) repeat steps (2) to (4) 35 more times; (6) 72° C. for 10minutes; (7) 4° C. hold.

The 50 μL PCR reactions were run on 1.5% agarose 1×TBE gels. Bands wereexcised from the gels and purified using the QIAquick Gel Extaction Kit(Qiagen, catalog #28706) according to the manufacturer's instructionsexcept that the DNA was eluted from the column using 35 μL of EB buffer.

The purified DNA was subcloned using the TOPO-TA cloning kit(Invitrogen, catalog #K4500-01) and the cloned inserts sequenced (by theDavis Sequencing Center, University of California at Davis, Calif.)using the M13 forward and M13 reverse primers. The resulting sequenceswere BLAST searched using the NCBI online search(http://www.ncbi.nlm.nih.gov/BLAST/) and aligned using the onlinesequence alignment tool ClustalW (http://www.ebi.ac.uk/clustalw/) todetermine base pair identity between the cloned insert and the genome.

The M. bovis clone was 1345 bp in length and perfectly matched bases259,504 to 260,858 of the M. bovis genome (Genbank accession numberNC_(—)002945.3), with the exception of 3 bases within the forward primersequence [SEQ ID NO: 61]. Bases 14 to 16 are CAT in the primer(TGAACCGCCCCGGCATGTCC; SEQ ID NO: 61) and TGA in the genomic sequence(TGAACCGCCCCGGTGAGTCC; SEQ ID NO: 64). The M. tuberculosis 313 bp clonematched bases 3,120,580 to 3,120,893 of Mycobacterium tuberculosis H37Rvgenome (Genbank accession number AL123456). Both of these sequences arewithin the Direct Repeat region of the genomes.

Example 33

This example illustrates the smartDNA reaction by PNA:RNA oligo hybridsin a non-denaturing PAGE gel.

RNA oligonucleotides (Sigma-Proligo) [SEQ ID NOS: 65-70, see Table 39]complementary to PNAs TB01-TB06 [SEQ ID NOS: 36-41, see Table 39],respectively, were diluted to 50 μM stocks with DEPC-treated molecularbiology grade, RNase-free water (Hyclone, catalog #SH30538.03).

TABLE 39 SEQ ID NO. Sequence Name 65 GUUUUGGGUCUGACGAC rTB01comp 66GUUUCCGUCCCCUCUCG rTB02comp 67 CAUGCCGGGGCGGUUCA rTB03comp 68UCGCCCGUCUACUUGGU rTB04comp 69 CGACCGACGGUUGGAUG rTB05comp 70CGGGAUGCAUGUCUUGU rTB06comp 36 n-biotin-(OO)-GTCGTCAGACCCAAAAC-c TB01 37n-biotin-(OO)-CGAGAGGGGACGGAAAC-c TB02 38n-biotin-(OO)-TGAACCGCCCCGGCATG-c TB03 39n-biotin-(OO)-ACCAAGTAGACGGGCGA-c TB04 40n-biotin-(OO)-CATCCAACCGTCGGTCG-c TB05 41n-biotin-(OO)-ACAAGACATGCATCCCG-c TB06

5 μL of 50 μM stock (250 picomoles) of each RNA oligo was mixed with 5μL of 50 μM stock (250 picomoles) of its complementary PNA in 1.5 mLmicrocentrifuge tubes (Eppendorf, catalog #022364111). The tubes wereincubated at room temperature for 10 minutes. Five μL of 25% glycerol(JT Baker, catalog #4043-00) was added to each sample.

The samples were loaded onto a 10% non-denaturing PAGE (19:1crosslinking; JT Baker, catalog #4968-00), 1×TBE gel. The gel was run at300 volts, for 45 minutes in 1×TBE gel running buffer. The gel wasstained with 15 μM DiSC₂(3) in 1×TBE for 30 minutes with gentleagitation. The reactions were activated using the Fritz Aurora 50/50fluorescent lamp (Fritz Industries, Inc., Mesquite, Tex.) for varyinglengths of time from T_(0min) to T_(3min). Photographs of the gel weretaken using a B&W Polaroid camera while the gel was illuminated with a254 nm UV transilluminator. Prior to photoactivation, the gel was pinkin color, with faint pink bands in lanes 1, 3, 5, 7, 9, 11 correspondingto the position of the PNA:RNA hybrids. Lanes 2, 4, 6, 8, 10, 12 alsohad faint bands corresponding to the position of the RNA oligos. Asexpected, the RNA:PNA hybrid migrated more slowly in the gel than theRNA oligos. During photoactivation, in lanes 1, 3, 5, 7, 9, 11 at theexpected location of the PNA:RNA hybrids, regions lacking colorappeared. These “holes” show up as darker areas on the second panel ofFIG. 23, after 3 minutes of light activation time.

Example 34

The specificity of the homogeneous smartDNA assay was tested using 2 ngof non-specific genomic DNAs and PNA TB 19: n-lys-TGAACCGCCCCGGCATG-c(SEQ ID NO: 71).

Samples containing genomic DNA isolated from several non-specificorganisms (see Table 40) were diluted to 2 ng per 25 μL in 1 mM EDTA.Aliquots of 25 μL of each of the DNA samples were dispensed in twotriplicate sets into a 384-white-well optical bottom microtiter plate(NUNC, catalog #242763). One set was the DNA only sample set thatcontained DNA and dye plus buffer sample. One set was the DNA+PNA samplethat contained DNA, PNA, dye plus buffer.

Buffer solution was prepared by dissolving 20 millimoles of homopipesbuffer (Research Organics catalog #6047H) in one liter of molecularbiology grade water (Hyclone, catalog #SH30538.03) and the pH wasadjusted to 5.0 with sodium hydroxide. Tween® 80 (Pierce Biotechnology,catalog #28328) was added to the final concentration of 0.1% to generate2×HP5.0 buffer. The resulting buffer solution was sterile filtered (MF75Vacuum Filter Units, PES Membrane, Nalgene, catalog #566-0020).

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) into2×HP5.0 buffer to a working concentration of 36 μM. PNA probe mixturewas prepared by diluting 50 μM PNA stock to 640 nM with 2×HP5.0 buffer.

After the DNA samples were dispensed into the microplate, two dyemixtures were then prepared. The reaction set-up steps involving the dyewere performed in a dimly lit room. The first control dye solution wasprepared by mixing equal volumes of 36 μM DiSC₂(3) in 2×HP5.0 bufferwith 2×HP5.0 buffer. This 18 μM DiSC₂(3) control solution was mixed byinversion in a 15 mL conical tube, and poured into a reagent reservoir.A 25 μL aliquot of the control dye solution was then dispensed to theDNA only samples in the 384-well plate. The second, dye+PNA probe mixwas prepared by mixing equal volumes of 36 μM DiSC₂(3) with the 640 nMPNA probe mix. This 18 μM DiSC₂(3)+320 nM PNA probe mix was mixed byinversion in a 15 mL conical tube, and poured into a reagent reservoir.A 25 μL aliquot of the dye+PNA probe mix was then dispensed to each DNAsample in the DNA+PNA sample set and gently shaken to mix the solutions.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 1 secondmedium-intensity orbital shake, followed by a 1 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 20 minutes total exposureto light.

The initial rates of dye photobleaching (initial dye color change rates)were calculated by subtracting the absorbance of the solution attime_(4min) from the initial absorbance of the solution at Time_(0min)and dividing by 4 minutes. Table 40 lists the initial dye color changerates depicted as change in milliabsorbance (mAbs) units per minute.

The PNA target sequence is absent from the human genome, severalbacteria that are present in or cause disease in humans as well as theplant Arabidopsis. From examining the initial dye color change rates ofthe smartDNA reactions for these organisms, the specificity of thisMTB-targeted DNA is apparent. We occasionally observed what appears tobe a specific dye color change reaction, as is observed for Arabidopsis,when a perfect match to the PNA probe is absent. We believe that thismaybe due to either the method of DNA isolation and purification oralternately the publicly available BLAST software was unable todetermine the presence of the PNA binding site in Arabidopsis becausethe length of the PNA is only 17 base pairs.

TABLE 40 standard initial deviation of dye color initial dye change ratecolor change (mAbs/min) rate DNA + DNA DNA + DNA DNA source catalog #PNA only PNA only Arabidopsis 42.05 14.11 3.82 0.05 thaliana E. coli17.13 14.50 1.27 1.30 human Sigma D7011 (lot 11.55 12.26 1.50 0.8302153787) human Sigma D3035 (lot 12.05 12.78 1.26 2.42 123K3796) P.aeruginosa ATCC 17933D 13.56 12.33 2.21 0.93 P. aeruginosa ATCC 47085D12.92 11.44 1.19 0.24 S. aureus ATCC 10832D 16.66 12.75 0.18 1.59 H.influenza ATCC 51907D 15.40 12.05 1.43 0.05 N. meningititis ATCCBAA-335D 11.92 9.55 1.32 0.79 M. microti 40.86 22.76 2.91 1.52 S.pneumoniae ATCC BAA-334D 11.50 9.62 0.47 0.04 K. pneumonia ATCC 700721D21.02 19.34 0.15 10.26 MTB 2 ng 58.25 12.10 4.05 0.17 MTB 1 ng 37.9811.92 0.26 0.50 MTB 0.5 ng 19.93 10.96 0.31 0.33 MTB 0.25 ng 14.42 10.430.23 1.00 MTB 0.125 ng 12.35 10.82 1.13 1.69 MTB 0.0625 ng 11.65 10.241.84 0.10 PNA only 9.81 9.47 0.78 1.09

Example 35

This example illustrates one embodiment of the present invention fordetermining the presence of DNA in a crude cell lysate in a homogenousassay.

Mycobacterium tuberculosis (MTB H37Ra, ATCC catalog #25117) cells weregrown in Middlebrook 7H9Broth with Tween® 80 (Remel Inc., catalog#R09556) were grown for 6 weeks at 37° C. with gentle agitation. Five mLof culture was diluted with 15 mL 1×PBS (Teknova, catalog #PO₂₀₀) plus 1mM EDTA to an equivalent of McFarland standard of 1 (˜1×10⁸ colonyforming units/mL). For each sample, 1 mL of cells was placed in a 1.5 mLmicrocentrifuge tube (Eppendorf, catalog #022364111) and centrifuged for1 minute at 14,000 rpm to pellet the cells. The supernatant was removedand the cells resuspended in 100 μL 1×PBS, 1 mM EDTA buffer. From aslurry of 0.1 mm glass beads (Scientific Industries, catalog #SI-BG01)and 1×PBS, 1 mM EDTA buffer, approximately 50 mg of beads were added toeach tube. The tubes were shaken at full speed for 2 minutes using theVortex Genie® 2 (Scientific Industries, catalog #SI-0236, model #G650)equipped with the TurboMix attachment for 1.5 mL microcentrifuge tubes(Scientific Industires, catalog #SI-0563). Following cell disruption,the tubes were placed on ice.

The concentration of DNA released from the cells was estimated by gelelectrophoresis. Twenty μL of each cell lysate was run on 0.8% agarose,1×TBE gel stained with ethidium bromide. As a standard for DNAconcentration, 2-fold dilutions of salmon sperm DNA were runside-by-side on the gel. The concentration of DNA in the cell lysateswas estimated by comparing the intensity of the bands of the sampleswith those of the standards.

Two-fold serial dilutions of the cell lysates were made using 1 mM EDTAto DNA concentrations of approximately 0.08 ng/μL; 0.04 ng/μL; 0.02ng/μL; 0.01 ng/μL. 25 μL of each DNA dilution was placed in triplicateinto a 384-white-well optical bottom microtiter plate (NUNC, catalog#242763) along with a no DNA control of 25 μL of 1 mM EDTA. In additionas a control, 2-fold dilutions of isolated MTB DNA was pipette intriplicate into the same 384-well microtiter plate.

Buffer solution was prepared by dissolving 20 millimoles of homopipesbuffer (Research Organics catalog #6047H) in one liter of molecularbiology grade water (Hyclone, catalog #SH30538.03) and the pH adjustedto 5.0 with sodium hydroxide. Tween® 80 (Pierce Biotechnology, catalog#28328) was added to the final concentration of 0.1% to generate 2×HP5.0buffer. The resulting buffer solution was sterile filtered (MF75 VacuumFilter Units, PES Membrane, Nalgene, catalog #566-0020).

Dye solution was prepared by diluting 7.5 mM3,3′-diethylthiacarbocyanine (“DiSC₂(3)”) (solubilized in DMSO) into2×HP5.0 buffer to a working concentration of 36 μM. PNA probe mixturewas prepared by diluting 50 μM PNA [TB 15, n-lys-ACCAAGTAGACGGGCGA-c(SEQ ID NO: 72)] stock to 640 nM with 2×HP5.0 buffer.

After the DNA samples were dispensed into the microplate, two dyemixtures were then prepared. The reaction set-up steps involving the dyewere performed in a dimly lit room. The first control dye solution wasprepared by mixing equal volumes of 36 μM DiSC₂(3) in 2×HP5.0 bufferwith 2×HP5.0 buffer. This 18 μM DiSC₂(3) control solution was mixed byinversion in a 15 mL conical tube, and poured into a reagent reservoir.A 25 μL aliquot of the control dye solution was then dispensed to theDNA only samples in the 384-well plate. The second, dye+PNA probe mixwas prepared by mixing equal volumes of 36 μM DiSC₂(3) with the 640 nMPNA probe mix. This 18 μM DiSC₂(3)+320 nM PNA probe mix was mixed byinversion in a 15 mL conical tube, and poured into a reagent reservoir.A 25 μL aliquot of the dye+PNA probe mix was then dispensed to each DNAsample in the DNA+PNA sample set and gently shaken to mix the solutions.

The microplate was inserted into the Tecan Safire² monochromator-basedmicroplate reader. The microplate was subjected to a 1 secondmedium-intensity orbital shake, followed by a 1 second settle time,followed by an absorbance measurement at 556 nm. The absorbancemeasurement was performed with a bandwidth of 20 nm, with 12 reads perwell. After the initial optical measurement, the plate was removed fromthe microplate reader, and subjected to photoactivation for a two minuteinterval. The light for photoactivation was provided by a solid-stateactivator providing illumination with a peak wavelength of 470 nm and apower density of approximately 1.8 mW/cm², as measured with alaser-based power meter having the tradename LaserCheck™ (Coherent,Inc., Santa Clara, Calif.; catalog #0217-271-00). The microplate wasreinserted into the reader and each well was measured as indicatedabove, however with a shortened orbital shake and settle time of onesecond each. This two minute photoactivation followed by absorbancedetection cycle was performed over a period of 20 minutes total exposureto light.

The initial rates of dye photobleaching (initial dye color change rates)were calculated by subtracting the absorbance of the solution attime_(4min) from the initial absorbance of the solution at Time_(0min)and dividing by 4 minutes. Table 41 lists the initial dye color changerates depicted as change in milliabsorbance (mAbs) units per minute.

The initial dye color change rates for both the DNA in the cell lysatesand the isolated DNA showed a dose response.

TABLE 41 2 ng 1 ng 0.5 ng 0.25 ng 0 ng initial dye color change rateaverages cell lysate alone 25.62 19.30 16.13 15.18 12.14 cell lysate +160 nM PNA 63.92 42.94 29.15 22.43 13.89 isolated DNA alone 15.37 14.2913.86 13.44 12.14 isolated DNA + 160 nM 77.25 61.68 45.66 34.37 13.89PNA standard deviations of initial dye color change rate cell lysatealone 0.64 0.54 1.13 0.81 0.44 cell lysate + 160 nM PNA 1.05 0.72 0.500.54 0.30 isolated DNA alone 0.63 0.70 0.96 0.66 0.44 isolated DNA + 160nM 6.65 2.71 2.69 1.04 0.30 PNA

Additional Embodiments

In one aspect, the invention relates to a method comprising the step ofcombining

-   -   (i) a sample that may or may not contain a target        polynucleotide;    -   (ii) a nucleic acid analog immobilized to a solid substrate,        wherein the nucleic acid analog is complementary to a portion of        the target polynucleotide; and    -   (iii) a light reactive dye capable of forming a light reactive        complex with the target polynucleotide and a complementary        nucleic acid analog,    -   wherein the target polynucleotide, if present, forms a light        reactive complex with the nucleic acid analog and the light        reactive dye.

The method may further comprise the step of isolating the solidsubstrate, based on a property of the solid substrate. The method mayfurther comprise the step of washing the solid substrate with a washsolution comprising the light reactive dye. The light reactive complexmay be formed at a room temperature. The light reactive complex mayalternatively be formed at a temperature of less than about 37° C. Thelight reactive complex may also be formed at a temperature of less thanabout 45° C. The light reactive dye may be selected from the groupconsisting of: 3,3′-diethylthiacarbocyanine,3,3′-diallylthiacarbocyanine, 3,3′-diethyl-9-methylthiacarbocyanine,3,3′-dibutylthiacarbocyanine, 3,3′-dipropylthiacarbocyanine,3,3′-dipentylthiacarbocyanine, and 1,1′-diethyl-2,2′-carbocyanine, andsalts thereof. The light reactive dye may be3,3′-Diethylthiacarbocyanine or salts thereof. The nucleic acid analogmay be less than 20 bases in length. The nucleic acid analog may beabout 17 bases in length. The nucleic acid analog may be selected fromthe group consisting of: PNA polynucleotides, LNA polynucleotides andmorpholino polynucleotides. The nucleic acid analog may be a PNApolynucleotide. The method may further comprise exposing the solidsubstrate to light under conditions sufficient to elute the targetpolynucleotide from the solid substrate. In the method, the step ofexposing the light reactive complex to light may be performed underambient light conditions. In the method, the step of exposing the lightreactive complex to light may be performed at ambient room temperature.In the method, the step of exposing the light reactive complex to lightmay be performed at a temperature of less than about 37° C. In themethod, the step of exposing the light reactive complex to light may beperformed at a temperature of less than about 45° C. In the method, thestep of exposing the light reactive complex to light may be performedfor a period of less than 1 hour. In the method, the step of exposingthe light reactive complex to light may be performed for a period ofless than 30 minutes. In the method, the step of exposing the lightreactive complex to light may be performed for a period of less than 5minutes. In the method, the light may include a controlled light. In themethod, the light may be from a light emitting diode (LED). In themethod, the majority of the light may have a specific wavelength ofbetween about 460 nm to about 480 nm.

In another embodiment, the invention relates to a method comprising thestep of combining

-   -   (i) a sample that may or may not contain a target molecule;    -   (ii) an immobilized polynucleotide bound to a solid substrate;    -   (iii) a bridging complex comprising (1) a polynucleotide portion        complementary to the immobilized polynucleotide, wherein at        least one of the immobilized polynucleotide and the        polynucleotide portion of the bridging complex is a nucleic acid        analog polynucleotide, and (2) a target-specific portion capable        of binding to the target molecule, wherein the bridging complex        comprises one or more molecules complexed together; and    -   (iv) a light reactive dye capable of forming a light reactive        complex with the immobilized polynucleotide and the        complementary polynucleotide portion of the bridging complex.

The method may further comprise the step of separating the solidsubstrate from the sample, based on a property of the solid substrate.The method may further comprise the step of washing the solid substratewith a wash solution comprising the light reactive dye. The lightreactive complex may be formed under ambient light conditions. The lightreactive complex may be formed at room temperature. The light reactivecomplex may be formed at a temperature of less than about 37° C. Thelight reactive complex may be formed at a temperature of less than about45° C. The light reactive dye may be selected from the group consistingof: 3,3′-diethylthiacarbocyanine, 3,3′-diallylthiacarbocyanine,3,3′-diethyl-9-methylthiacarbocyanine, 3,3′-dibutylthiacarbocyanine,3,3′-dipropylthiacarbocyanine, 3,3′-dipentylthiacarbocyanine, and1,1′-diethyl-2,2′-carbocyanine, and salts thereof. The light reactivedye may be 3,3′-Diethylthiacarbocyanine or salts thereof. The nucleicacid analog may be less than 20 bases in length. The nucleic acid analogmay be about 17 bases in length. The nucleic acid analog may be selectedfrom the group consisting of: PNA polynucleotides, LNA polynucleotidesand morpholino polynucleotides. The nucleic acid analog may be a PNApolynucleotide. The method may further comprise the step of detectingthe relative change in an optical property of the light reactivecomplex. The method may further comprise exposing the solid substrate tolight under conditions sufficient to elute the target molecule from thesolid substrate. In the method, the step of exposing the light reactivecomplex to light may be performed under ambient light conditions. In themethod, the step of exposing the light reactive complex to light may beperformed at ambient room temperature. In the method, the step ofexposing the light reactive complex to light may be performed at atemperature of less than about 37° C. In the method, the step ofexposing the light reactive complex to light may be performed at atemperature of less than about 45° C. In the method, the step ofexposing the light reactive complex to light may be performed for aperiod of less than 2 hours. In the method, the step of exposing thelight reactive complex to light may be performed for a period less than1 hour. In the method, the step of exposing the light reactive complexto light may be performed for a period less than 30 minutes. In themethod, the step of exposing the light reactive complex to light may beperformed for a period less than 5 minutes. In the method, the targetmolecule may be a polypeptide and the target-specific portion of thebridging complex is a polypeptide binding molecule capable ofspecifically binding the polypeptide target molecule. In the method, thetarget molecule may be a polypeptide and the target-specific portion ofthe bridging complex may be an antibody or binding fragment thereofcapable of specifically binding the polypeptide target molecule. In themethod, the target molecule may be an oligosaccharide moiety and thetarget-specific portion of the bridging complex may be selected from thegroup consisting of an antibody or binding fragment thereof or a lectinmolecule, capable of specifically binding the polypeptide targetmolecule. In the method, the target molecule may be a hapten moleculeand the target-specific portion of the bridging complex may be anantibody or binding fragment thereof capable of specifically binding thehapten target molecule. In the method, the target molecule may be apolynucleotide and the target-specific portion of the bridging complexmay be a nucleic acid analog polynucleotide complementary to thepolynucleotide target molecule.

In one other aspect, the invention relates to a method for making anucleic acid diagnostic kit comprising the step of combining, in apackaged combination,

-   -   (i) an immobilized nucleic acid analog bound to a solid        substrate, wherein the nucleic acid analog is complementary to a        portion of a target polynucleotide; and    -   (ii) a light reactive dye in a container, wherein the light        reactive dye is capable of forming a light reactive complex with        the target polynucleotide and a complementary nucleic acid        analog.    -   In yet further embodiment, the invention relates to a method for        making a nucleic acid diagnostic kit comprising the step of        combining, in a packaged combination,    -   (i) an immobilized polynucleotide bound to a solid substrate;        and    -   (ii) a container comprising at least one of:        -   a bridging complex comprising (1) a polynucleotide portion            complementary to the immobilized polynucleotide, wherein at            least one of the immobilized polynucleotide and the            polynucleotide portion of the bridging complex is a nucleic            acid analog polynucleotide, and (2) a target-specific            portion capable of binding to the target molecule, wherein            the bridging complex comprises one or more molecules            complexed together; and        -   a light reactive dye capable of forming a light reactive            complex with the immobilized polynucleotide and the            complementary polynucleotide portion of the bridging            complex;

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entireties, respectively, forall purposes and to the same extent as if each individual publication,patent, or patent application were specifically and individuallyindicated to be so incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it is readily apparentto those of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit and scope of the disclosure.

1. A method comprising (a) combining (i) a sample that may or may not contain a target polynucleotide; (ii) a nucleic acid analog immobilized to a solid substrate, wherein the nucleic acid analog is complementary to at least a portion of the target polynucleotide; (iii) a light reactive dye capable of forming a light reactive complex with the target polynucleotide and a complementary nucleic acid analog, wherein the target polynucleotide, if present, forms a light reactive complex with the nucleic acid analog and the light reactive dye; and (b) exposing the nucleic acid analog immobilized to the solid substrate to light under conditions sufficient to elute the target polynucleotide from the solid substrate.
 2. The method of claim 1, wherein in the step of exposing the light reactive complex to light, the light used comprises ambient light.
 3. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed at ambient room temperature.
 4. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed at a temperature of less than about 37° C.
 5. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed at a temperature of less than about 45° C.
 6. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed for a period of less than 1 hour.
 7. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed for a period of less than 30 minutes.
 8. The method of claim 1, wherein the step of exposing the light reactive complex to light is performed for a period of less than 5 minutes.
 9. The method of claim 1, wherein the light comprises a controlled light.
 10. The method of claim 1, wherein the light is from a light emitting diode.
 11. The method of claim 1, wherein the majority of the light has a specific wavelength of between about 460 nm to about 480 nm.
 12. A method comprising the steps of (a) combining (i) a sample that may or may not contain a target molecule; (ii) an immobilized polynucleotide bound to a solid substrate; (iii) a bridging complex comprising (1) a polynucleotide portion complementary to the immobilized polynucleotide, wherein at least one of the immobilized polynucleotide and the polynucleotide portion of the bridging complex is a nucleic acid analog polynucleotide, and (2) a target-specific portion capable of binding to the target molecule, wherein the bridging complex comprises one or more molecules complexed together; and (iv) a light reactive dye capable of forming a light reactive complex with the immobilized polynucleotide and the complementary polynucleotide portion of the bridging complex, wherein the target molecule, if present in the sample, binds to the target-specific portion of the bridging complex; and (b) exposing the immobilized polynucleotide bound to the solid substrate to light under conditions sufficient to elute the target polynucleotide from the solid substrate.
 13. The method of claim 1, further comprising the step of isolating the solid substrate, based on a property of the solid substrate.
 14. The method of claim 1, further comprising the step of washing the solid substrate with a wash solution comprising the light reactive dye.
 15. The method of claim 1, wherein the light reactive complex is formed at a room temperature.
 16. The method of claim 1, wherein the light reactive complex is formed at a temperature of less than about 37° C.
 17. The method of claim 1, wherein the light reactive complex is formed at a temperature of less than about 45° C.
 18. The method of claim 1, wherein the light reactive dye is selected from the group consisting of: 3,3′-diethylthiacarbocyanine, 3,3′-diallylthiacarbocyanine, 3,3′-diethyl-9-methylthiacarbocyanine, 3,3′-dibutylthiacarbocyanine, 3,3′-dipropylthiacarbocyanine, 3,3′-dipentylthiacarbocyanine, and 1,1′-diethyl-2,2′-carbocyanine, and salts thereof.
 19. The method of claim 1, wherein the light reactive dye is 3,3′-diethylthiacarbocyanine or salts thereof.
 20. The method of claim 1, wherein the nucleic acid analog is less than 20 bases in length.
 21. The method of claim 1, wherein the nucleic acid analog is about 17 bases in length.
 22. The method of claim 1, wherein the nucleic acid analog is about 15 bases in length.
 23. The method of claim 1, wherein the nucleic acid analog is selected from the group consisting of: PNA polynucleotides, LNA polynucleotides and morpholino polynucleotides.
 24. The method of claim 1, wherein the nucleic acid analog is a PNA polynucleotide. 